Solution structure of the cyclotide palicourein: Implications for the development of a pharmaceutical framework

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.

Designing supramolecular structures from models of cyclic peptide scaffolds with heterocyclic constraints

Cyclic peptides containing oxazole and thiazole heterocycles have been examined for their capacity to be used as scaffolds in larger, more complex, protein-like structures. Both the macrocyclic scaffolds and the supramolecular structures derived therefrom have been visualised by molecular modelling techniques. These molecules are too symmetrical to examine structurally by NMR spectroscopy. The cyclic hexapeptide ([Aaa-Thz](3), [Aaa-Oxz](3)) and cyclic octapeptide ([Aaa-Thz](4), [Aaa-Oxz](4)) analogues are composed of dipeptide surrogates (Aaa: amino acid, Thz: thiazole, Oxz: oxazole) derived from intramolecular condensation of cysteine or serine/threonine side chains in dipeptides like Aaa-Cys, Aaa-Ser and Aaa-Thr. The five-membered heterocyclic rings, like thiazole, oxazole and reduced analogues like thiazoline, thiazolidine and oxazoline have profound influences on the structures and bioactivities of cyclic peptides derived therefrom. This work suggests that such constrained cyclic peptides can be used as scaffolds to create a range of novel protein-like supramolecular structures (e.g. cylinders, troughs, cones, multi-loop structures, helix bundles) that are comparable in size, shape and composition to bioactive surfaces of proteins. They may therefore represent interesting starting points for the design of novel artificial proteins and artificial enzymes. (C) 2002 Elsevier Science Inc. All rights reserved.

Small molecules that mimic components of bioactive protein surfaces

Small molecules designed to mimic specific structural components of a protein (peptide strands, sheets, turns, helices, or amino acids) can be expected to display agonist or antagonist biological responses by virtue of interacting with the same receptors that recognize the protein. Here we describe some minimalist approaches to structural mimetics of amino acids and of strand, turn, or helix segments of proteins. The designed molecules show potent and selective inhibition of protease, transferase, and phospholipase enzymes, or antagonism of G-protein coupled or transcriptional receptors, and have potent anti-tumour, anti-inflammatory, or antiviral activity.

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the alpha-conotoxin MII

Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the alpha-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xerl oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides

Background: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. Results: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. Conclusion: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames.

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Peptide bundles: Loops, helices, strands and sheets

Protein-protein interactions are central to all biological processes. The creation of small molecules that can structurally mimic the fundamental units of protein architecture (helices, strands, turns, and their combinations) could potentially be used to reproduce important bioactive protein surfaces and interfere in biological processes. Although this field is still in relative infancy, substantial progress is being made in creating small molecules that can mimic these individual secondary structural elements of proteins. However the generation of compounds that can reproduce larger protein surfaces, composed of multiple structural elements of proteins, has proven to be much more challenging. This presentation will describe some densely functionalised small molecules that do constrain multiple peptide motifs in defined structures such as loop bundles, helix bundles, strand and sheet bundles. An example of a helix bundle that undergoes conformational changes to a beta sheet bundle and aggregates into multi-micron length peptide nanofibre 'rope' will be described.

p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

A novel method for selection of chymotrypsin inhibitors from a phage peptide library

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

alpha-conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors

We have isolated and characterized ol-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus, The peptide was classified as an cy-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has ho mology to sequences of previously described cu-conotoxins, particularly PnIA, PnIB, and ImI, However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry, Native EpI was shown to coelute with synthetic EpI, The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides, The activities of synthetic EpI and its unsulfated analogue [Tyr(15)]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors), Both EpI and [Tyr(15)]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia, These results indicate that EPI and [Tyr(15)]EpI selectively inhibit alpha 3 beta 2 and alpha 3 beta 4 nicotinic acetylcholine receptors.

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands, As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, A beta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles, In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of A beta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6.0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for A beta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of A beta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the Literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

Solution structure of methionine-oxidized amyloid beta-peptide (1-40). Does oxidation affect conformational switching?

The solution structure of A beta(1-40)Met(O), the methionine-oxidized form of amyloid beta-peptide A beta(1-40), has been investigated by CD and NMR spectroscopy. Oxidation of Met35 may have implications in the aetiology of Alzheimer's disease. Circular dichroism experiments showed that whereas A beta(1-40) and A beta(1-40)Met(O) both adopt essentially random coil structures in water (pH 4) at micromolar concentrations, the former aggregates within several days while the latter is stable for at least 7 days under these conditions. This remarkable difference led us to determine the solution structure of A beta(1-40)Met(O) using H-1 NMR spectroscopy. In a water-SDS micelle medium needed to solubilize both peptides at the millimolar concentrations required to measure NMR spectra, chemical shift and NOE data for A beta(1-40)Met(O) strongly suggest the presence of a helical region between residues 16 and 24. This is supported by slow H-D exchange of amide protons in this region and by structure calculations using simulated annealing with the program XPLOR. The remainder of the structure is relatively disordered. Our previously reported NMR data for A beta(1-40) in the same solvent shows that helices are present over residues 15-24 (helix 1) and 28-36 (helix 2), Oxidation of Met35 thus causes a local and selective disruption of helix 2. In addition to this helix-coil rearrangement in aqueous micelles, the CD data show that oxidation inhibits a coil-to-beta-sheet transition in water. These significant structural rearrangements in the C-terminal region of A beta may be important clues to the chemistry and biology of A beta(1-40) and A beta(1-42).

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.