Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

Solution structure of Alo-3: a new knottin-type antifungal peptide from the insect Acrocinus longimanus

Insect peptides are key elements of the innate immunity against bacteria and fungi. These molecules offer remarkable properties: high efficacy, a low probability of resistance, limited toxicity, and immunogenicity. In this context, we are investigating several classes of peptides, and we have been successful in identifying biologically important classes of peptides and small molecules that will provide a stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and other pathologies of high medical need. Recently, we have isolated a new class of antifungal peptides from the coleopteran Acrocinus longimanus. Three homologous peptides, Alo-1, Alo-2, and Alo-3, with sequence identity above 80% and active against the Candida glabrata yeast strain were identified. Alo-3 displayed the highest activity against Candida glabrata and was thus chosen for structure determination using NMR spectroscopy and molecular modeling. Alo-3 contains six cysteine residues forming three disulfide bridges. The pairing of the cysteines was assessed using ambiguous disulfide restraints within the ARIA software, allowing us to establish that Alo-3 belongs to the inhibitor cystine-knot family. It exhibits all the structural features characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first strand to the second strand and a series of turns. To our knowledge, Alo-3 is the first peptide from insects with antimicrobial activity adopting the knottin fold. Alo-3 shows a level of activity significantly higher against C. glabrata than Alo-1 or Alo-2. It has no negatively charged residues and displays on its surface a cationic pole that may account for its antifungal activity. This finding is validated by the comparison of the structure of Alo-3 with the structure of other structurally related peptides from other sources also showing antifungal activity.     

Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein

Background

The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.

Methodology/Principal Findings

Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α_vβ₃ and α_vβ₅ integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α₅β₁ and α_iibβ₃. In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α_vβ₃ and α_vβ₅ integrins. A ⁶⁴Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3–5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.

Conclusions/Significance

We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.     

Structural analysis of free and enzyme-bound amaranth alpha-amylase inhibitor: classification within the knottin fold superfamily and analysis of its functional flexibility.

The three-dimensional structure of the amaranth alpha-amylase inhibitor (AAI) adopts a knottin fold of abcabc topology. Upon binding to alpha-amylase, it adopts a more compact conformation characterized by an increased number of intramolecular hydrogen bonds, a decreased volume and in addition a trans to cis isomerization of Pro20. A systematic analysis of the 3-D structural databanks revealed that similar proteins and domains share with AAI the characteristic presence of proline residues, many of which are in a cis backbone conformation. As these proteins fulfil a variety of functional roles and are expressed in very different organisms, we conclude that the structure of the knottin fold, including the propensity of the cis bond, are the result of convergent evolution.     

Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.     

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC₅₀-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.     

Solution structure of termicin,an antimicrobial peptide from the termite Pseudacanthotermes spiniger

The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine-rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an alpha-helical segment (Phe4-Gln14) and a two-stranded (Phe19-Asp25 and Gln28-Phe33) antiparallel beta-sheet forming a "cysteine stabilized alphabeta motif" (CSalphabeta) also found in antibacterial and antifungal defensins from insects and from plants. Interestingly, termicin shares more structural similarities with the antibacterial insect defensins and with MGD-1, a mussel defensin, than with the insect antifungal defensins such as drosomycin and heliomicin. These structural comparisons suggest that global fold alone does not explain the difference between antifungals and antibacterials. The antifungal properties of termicin may be related to its marked hydrophobicity and its amphipatic structure as compared to the antibacterial defensins. [SWISS-PROT accession number: Termicin (P82321); PDB accession number: 1MM0.]     

Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.     

cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthia aurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5′-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Ciona intestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (−) bacteria, while evidencing no hemolytic activity toward human erythrocytes.     

Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1–19) and Pro (aa 20–53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.     

NMR solution structure of ATTp,an Arabidopsis thaliana trypsin inhibitor

The three-dimensional structure of the precursor form of the Arabidopsis thaliana trypsin inhibitor (ATT(p), GenBank entry Z46816), a 68-residue (approximately 7.5 kDa) rapeseed class proteinase inhibitor, has been determined in solution at pH 5.0 and 25 degrees C by multinuclear magnetic resonance spectroscopy. The protein contains one alpha-helix and two strands of antiparallel beta-sheet, with a type IV beta-turn connecting the two strands. The alpha-helix and the inhibitory loop are connected to the beta-sheet through three disulfide bridges; a fourth disulfide bridge connects the N- and C-termini. The overall structural topology of ATT(p) is similar to those of the sweet tasting protein brazzein (rmsd of 3.0 A) and the antifungal protein Rs-Afp1 [a knottin protein from radish (Raphanus sativus), rmsd of 2.7 A]. The precursor segment in ATT(p) is disordered, as visualized by the final 20-conformer ensemble and as confirmed by (15)N heteronuclear NOE analysis. The overall fold of ATT(p) is distinct from those of other classes of serine proteinase inhibitors except in the inhibitor loop; therefore, it represents a new inhibitor fold.     

Antifungal activity of a de novo synthetic peptide and derivatives against fungal food contaminants

The development of novel solutions to fight microbial food contaminants rests upon two pillars, which are the development of resistant strains and consumers' desire for a reduced consumption of synthetic drugs. Natural antimicrobial peptides possess the qualities to overcome these issues. De novo synthesis of novel antifungal compounds is a major progress that has been facilitated by the identification of parameters involved in the antimicrobial activity. A 14‐residue peptide named KK14, with the sequence KKFFRAWWAPRFLK‐NH₂, was designed and inhibited conidial germination and fungal growth of food contaminants within the range 6.25 to 50 μg/ml and 6.25 to 100 μg/ml, respectively. The study of three analogues of the peptide highlighted the role of some residues in the structural conformation of the peptide and its antifungal activity. The substitution of a Pro residue with Arg increased the helical content of the peptide not only its antifungal activity but also its cytotoxicity. The insertion of an unnatural bulky residue β‐diphenylalanine or a full d ‐enantiomerization overall increased the antifungal potency. The four peptides showed similar behaviour towards salt increase, heat treatment, and pH decrease. Interestingly, the d enantiomer remained the most active at high pH and after proteolytic digestion. The four peptides did not present haemolytic activity up to 200 μg/ml but had different behaviours of cytotoxicity. These differences could be crucial for potential application as pharmaceutical or food preservatives.     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Fungicidal effect of pleurocidin by membrane-active mechanism and design of enantiomeric analogue for proteolytic resistance

Pleurocidin (Ple) is a 25-residue peptide which is derived from the skin mucous secretion of the winter flounder (Pleuronectes americanus). In this study, we investigated antifungal effects and its mode of action of Ple on human pathogenic fungi. Ple showed potent antifungal activity with low hemolytic activity. To investigate the antifungal mechanisms of Ple, the cellular localization and membrane interaction of Ple were examined. Protoplast regeneration and membrane-disrupting activity by DPH-labeled membrane support the idea, that Ple exerts fungicidal activity against the human pathogenic fungus Candida albicans with the disruption of a plasma membrane. To aim for which was the application of a therapeutic agent, we designed a synthetic enantiomeric peptide composed of all-d-amino acids to enhance proteolytic resistance. The synthetic all-d-Ple also displayed two-fold more potent antifungal activity than that of all-l-Ple, and its antifungal activity showed proteolytic resistance against various proteases. Therefore, these results suggest a therapeutic potential of all-d-Ple with regard to its proteolytic resistance against human fungal infections.     

Rational design and improvement of the dimerization‐disrupting peptide selectivity between ROCK‐I and ROCK‐II kinase isoforms in cerebrovascular diseases

Human rho‐associated coiled‐coil forming kinases (ROCKs) ROCK‐I and ROCK‐II have been documented as attractive therapeutic targets for cerebrovascular diseases. Although ROCK‐I and ROCK‐II share a high degree of structural conservation and are both present in classic rho/ROCK signaling pathway, their downstream substrates and pathological functions may be quite different. Selective targeting of the two kinase isoforms with traditional small‐molecule inhibitors is a great challenge due to their surprisingly high homology in kinase domain (~90%) and the full identity in kinase active site (100%). Here, instead of developing small‐molecule drugs to selectively target the adenosine triphosphate (ATP) site of two isoforms, we attempt to design peptide agents to selectively disrupt the homo‐dimerization event of ROCK kinases through their dimerization domains which have a relatively low conservation (~60%). Three helical peptides H1, H2, and H3 are split from the kinase dimerization domain, from which the isolated H2 peptide is found to have the best capability to rebind at the dimerization interface. A simulated annealing (SA) iteration method is used to improve the H2 peptide selectivity between ROCK‐I and ROCK‐II. The method accepts moderate degradation in peptide affinity in order to maximize the affinity difference between peptide binding to the two isoforms. Consequently, hundreds of parallel SA runs yielded six promising peptide candidates with ROCK‐I over ROCK‐II (I over II [IoII]) calculated selectivity and four promising peptide candidates with ROCK‐II over ROCK‐I (II over I [IIoI]) calculated selectivity. Subsequent anisotropy assays confirm that the selectivity values range between 13.2‐fold and 83.9‐fold for IoII peptides, and between 5.8‐fold and 21.2‐fold for IIoI peptides, which are considerably increased relative to wild‐type H2 peptide (2.6‐fold for IoII and 2.0‐fold for IIoI). The molecular origin of the designed peptide selectivity is also analyzed at structural level; it is revealed that the peptide residues can be classified into conserved, non‐conserved, and others, in which the non‐conserved residues play a crucial role in defining peptide selectivity, while conserved residues confer stability to kinase‐peptide binding.     

蜡螟-单梗着色霉Fonsecaea monophora感染模型的构建及其血淋巴功能的变化

暗色真菌单梗着色霉Fonsecaea monophora侵犯皮肤及皮下组织,引起着色芽生菌病;其所引起的皮肤疣状改变或溃疡严重影响患者生活质量,重者可致残甚至发生癌变。近年来,无脊椎动物构建感染模型逐渐兴起,应用范围逐渐扩大。本研究利用蜡螟成功构建了F. monophora体内感染的动物模型,绘制了被感染蜡螟幼虫的生存曲线,观察记录了感染过程中蜡螟幼虫体表及体腔内的变化情况。在此基础上,进一步探索蜡螟幼虫对F. monophora的免疫防御应答,初步研究发现由病原体感染所诱发的血淋巴改变具有一定的抑真菌性,可抑制白念珠菌的生长。     

水稻穗长基因PAL3的克隆及自然变异分析

穗型是决定水稻(Oryza sativa)产量的关键因素之一。我们从粳稻品种圣稻808 (SD808)的EMS诱变突变体库中发现4份短穗突变体,这些突变体的穗长、一级枝梗数、二级枝梗数和穗粒数发生不同程度的降低。基因定位和图位克隆表明,这些突变体的表型受同一基因控制,将该基因命名为PAL3 (PANICLE LENGT...