The solution structure of a copper(II) compound of a new cyclic octapeptide by EPR spectroscopy and force field calculations

A new cyclic octapeptide, cyclo(Ile-Ser-(Gly)Thz-Ile-Thr-(Gly)Thz) (PatN), related to patellamide A, has been synthesized and reacted with copper(II) and base to form mono- and dinuclear complexes. The coordination environments around copper(TI) have been characterized by EPR spectroscopy. The solution structure of the thermodynamically most stable product, a purple dicopper(TI) compound, has been examined by simulating weakly dipole-dipole coupled EPR spectra based upon structural parameters obtained from force field (MM and MD) calculations. The MM-EPR method produces a saddle-shaped structure for [Cu-2(PatN)(OH2)(6)] that is similar to the known solution structure of patellamide A and the known solid-state structure of [Cu-2(AscidH(2))CO3(OH2)(2)]. Compared with the latter, [Cu-2(PatN)] has no carbonate bridge and a significantly flatter topology. The MM-EPR approach to solution-structure determination for paramagnetic metallopeptides may find wide applications to other metallopeptides and metalloproteins.

Solution structure of a defensin-like peptide from platypus venom

Three defensin-like peptides (DLPs) were isolated from platypus venom and sequenced. One of these peptides, DLP-1, was synthesized chemically and its three-dimensional structure was determined using NMR spectroscopy. The main structural elements of this 42-residue peptide were an anti-parallel beta-sheet comprising residues 15-18 and 37-40 and a small 3(10) helix spanning residues 10-12. The overall three-dimensional fold is similar to that of beta-defensin-12, and similar to the sodium-channel neurotoxin ShI (Stichodactyla helianthus neurotoxin I). However, the side chains known to be functionally important in beta-defensin-12 and ShI are not conserved in DLP-1, suggesting that it has a different biological function. Consistent with this contention, we showed that DLP-1 possesses no anti-microbial properties and has no observable activity on rat dorsal-root-ganglion sodium-channel currents.

Increased ovulation rate in Merino ewes immunized against small synthetic peptide fragments of the inhibin alpha subunit

Four experiments were carried out in Merino ewes during a period of 4 years to determine the long-term effects of immunization against different synthetic peptides mimicking the amine terminal of the or subunit of porcine inhibin. Peptides were conjugated to human serum albumin and 100-200 mu g emulsified in Freund's complete adjuvant for the primary immunization. Usually two booster injections were given at monthly intervals with 50-100 mu g conjugated peptide using either incomplete Freund's adjuvant or Montanide : Marcel. In some experiments a further immunization was carried in the next year. Blood samples were taken 10 days after each immunization, during the luteal phase, for estimation of gonadotrophin concentrations and determination of inhibin antibody titres. One day after blood sampling cloprostenol was used to induce luteolysis and laparoscopy was performed in the subsequent oestrous cycle. Immunization of ewes with synthetic peptides 1-32, 1-26, 7-26 and 8-30 resulted in large increases in the ovulation rate (OR). An approximately two-fold increase in OR was observed following the first booster immunization with these peptides and a three- to five-fold increase after the second booster immunization. Immunization with these large peptides resulted in a sustained increase in OR for a period of at least 1 year after the second booster immunization. Of the shorter peptides, peptides 10-26 and 13-26 gave a reasonable ovulatory response, although it was more difficult to obtain a response with peptides 1-16, 8-22, 13-25, 8-19 and 10-19; peptides 7-13 and 1-6 gave no response (but were examined for one breeding season only). The smaller peptides led to lower inhibin antibody titres that were not necessarily associated with increased follicle-stimulating hormone (FSH) or OR. More intensive blood sampling in one experiment showed that following primary immunization against peptide 1-32 there was a transient increase in plasma FSH which did not lead to an increased OR. Moreover, a prolonged period of raised FSH after the first booster was significantly correlated with increased OR. In these animals antibody titres were only slightly increased after primary immunization, but after the first booster immunization higher titres were observed that were significantly correlated with trough FSH values and the subsequent OR. These results are interpreted as showing that (1) to obtain an increase in OR peptides 1-32, 1-26 and 7-26 are suitable as immunogens; (2) smaller peptides are less reliable, often require multiple injections, and the response may be delayed; and (3) an extended period of raised plasma FSH is needed to give a large ovulatory response.

Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Several macrocyclic peptides (similar to 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional struc ture of one of these novel peptides, cycloviolacin O1, using H-1 NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants. (C) 1999 Academic Press.

Conformations of cyclic octapeptides and the influence of heterocyclic ring constraints upon calcium binding

A comparison is made between the structures and calcium binding properties of four cyclic octapeptides that differ in the number of heterocyclic thiazole and oxazoline ring constraints. The conformations of the naturally occurring cyclic octapeptides ascidiacyclamide 1 and patellamide D 2, which each contain two oxazoline and two thiazole rings, are compared by H-1 NMR spectroscopy with the analogues cyclo(Thr-D-Val(Thz)-Ile)(2) 3 with just two thiazoles, and cyclo(Thr-D-Val-alpha Abu-Ile)(2) 4, with no 5-membered rings. The conformations observed in the solid state for ascidiacyclamide (saddle) and patellamide D (twisted figure of eight) were retained in solution, whilst peptide 3 was found to have a chair shape and peptide 4 displayed a range of conformations. The solid state structure of 4 revealed that the peptide takes a relatively planar conformation with a number of transannular hydrogen bonds, which are apparently retained in solution. Complexation studies utilising H-1 NMR and CD spectroscopy yielded 1∶1 calcium-peptide binding constants (log K) for the four peptides (2.9 (1), 2.8 (2), 4.0 (3) and 5.5 (4)) as well as a 1 : 2 metal-peptide binding constant for 3 (log K = 4.5). The affinity for Ca2+ thus decreases with increasing number of 5-membered ring constraints in the macrocycle (4 > 3 > 2 approximate to 1).

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

Cyclic octapeptides containing thiazole. Effect of stereochemistry and degree of flexibility on calcium binding properties

Solution conformation and calcium binding properties have been investigated for the two cyclic octapeptides cyclo(-D-Thr-D-Val(Thz)-Ile-)(2) (4) and cyclo(-Thr-Gly(Thz)-Ile-Ser-Gly(Thz)-Ile-)(5) and the results are compared to those for the cyclic octapeptides previously studied; ascidiacyclamide (1), patellamide D (2), cyclo(-Thr-D-Val(Thz)-Ile-)(2) (3), and cyclo(-Thr-D-Val-alphaAbu-Ile-)2 (6). Both 4 and 5 contain two heterocyclic thiazole ring constraints but the latter has a larger degree of flexibility as a consequence of the glycine residues within the cyclic framework. The solution conformation of 4 and 5 was determined from H-1 NMR spectra and found to be a twisted figure of eight similar to that for 2. Complexation studies using H-1 NMR and CD spectroscopy yielded 1 : 1 calcium-peptide binding constants (logK) for the two peptides (2.3 (4) and 5.7 (5)). For 5 the magnitude of the binding constant was verified by a competition titration using CD. The different calcium-binding affinities of 3 (logK = 4.0) and 4 is attributed to the stereochemistry of the threonine residue. The magnitude of the binding constant for 5 compared to 3 and 4 (all peptides containing two thiazole ring constrains) demonstrates that the increase in flexibility of the cyclic peptide has a dramatic effect on the Ca2+ binding ability. The affinity for Ca2+ thus decreases in the order (6 similar to 5 > 3 > 2 similar to 1 > 4). The number of carbonyl donors available on each peptide has only a limited effect on calcium binding. The most important factor is the flexibility, which allows for a conformation of the peptide capable of binding calcium efficiently.

Conformations of platypus venom C-type natriuretic peptide in aqueous solution and sodium dodecyl sulfate micelles

Nuclear magnetic resonance spectroscopy was used to investigate the conformations of the platypus venom C-type natriuretic peptide A (OvCNPa) in aqueous solutions and in solutions containing sodium dodecyl sulfate (SDS) micelles. The chemically synthesized OvCNPa showed a substantial decrease in flexibility in aqueous solution at 10 degreesC, allowing the observation of medium- and long-range nuclear Overhauser enhancement (NOE) connectivities. Three-dimensional structures calculated using these data showed flexible and reasonably well-defined regions, the locations of which were similar in the two solvents. In aqueous solution, the linear part that spans residues 3-14 was basically an extended conformation while the cyclic portion, defined by residues 23-39, contained a series of beta-turns. The overall shape of the cyclic portion was similar to that observed for an atrial natriuretic peptide (ANP) variant in aqueous solution. OvCNPa adopted a different conformation in SDS micelles wherein the N-terminal region, defined by residues 2-10, was more compact, characterised by turns and a helix, while the cyclic region had turns and an overall shape that was fundamentally different from those structures observed in aqueous solution. The hydrophobic cluster, situated at the centre of the ring of the structure in aqueous solution, was absent in the structure in the presence of SDS micelles. Thus, OvCNPa interacts with SDS micelles and can possibly form ion-channels in cell membranes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Interaction of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes, and its open chain analogue with membrane mimics

In contrast to other mammalian defensins, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue, oRTD-1, although both peptides adopt very similar structures in water. It was suggested that the additional charges at the termini of oRTD-1 are the cause for its lower antimicrobial activity. Therefore, we studied the interaction of both peptides with membrane mimics composed of zwitterionic (PC) and negatively charged (PG) phospholipids, major lipid components of erythrocyte and bacterial cell membranes, respectively. Microcalorimetry showed that RTD-1 and oRTD-1 did not affect the phase behavior of PC liposomes, while in PG liposomes both peptides induced new phase transitions above the chain melting transition of the lipid. The shape and fraction differed between both peptides, depending also on their concentration, which will be discussed in terms of their antimicrobial activity.

Inhibitors of TACE and Caspase-1 as anti-inflammatory drugs

TNF-alpha neutralising agents such as Infliximab (Remicade(R)), Etanercept (Enbrel(R)) and the IL-1 receptor antagonist Anakinra (Kineret(R)), are currently used clinically for the treatment of many inflammatory diseases such as Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis and psoriasis. These protein preparations are expensive to manufacture and administer, need to be injected and can cause allergic reactions. An alternative approach to lowering the levels of TNF-alpha and IL-1 beta in inflammatory disease, is to inhibit the enzymes that generate these cytokines using cheaper small molecules. This paper is a broad overview of the progress that has been achieved so far, with respect to small molecule inhibitor design and pharmacological studies (in animals and humans), for the metalloprotease Tumour Necrosis Factor-alpha Converting Enzyme (TACE) and the cysteine protease Caspase-1 (Interieukin-1 beta Converting Enzyme, ICE). Inhibitors of these two enzymes are currently considered to be good therapeutic targets that have the potential to provide relatively inexpensive and orally bioavailable anti-inflammatory agents in the future.

Zipping up transcription factors: Rational design of anti-Jun and anti-Fos peptides

Various members of the bZip and bHLH-Zip families of eukaryotic transcription factors, including Jun, Fos, and Myc, have been identified as oncoproteins; mutation or deregulated expression of these proteins leads to certain types of cancer. These proteins can only bind to their cognate DNA enhancer sites following homodimerization, or heterodimerization with another family member, via their leucine zipper domain. Thus, a novel anticancer strategy would be to inhibit dimerization of these proteins, thereby blocking their DNA binding and transactivation functions. In this paper we show that it is possible to rationally design leucine zipper peptides that bind with high affinity to the leucine zipper dimerization domains of c-Jun and c-Fos, thus preventing the formation of functional c-Jun homodimers and c-Jun:c-Fos heterodimers; we refer to such peptides as superzippers (SZs). In vivo, c-Jun:SZ and c-Fos:SZ heterodimers should be nonfunctional as they lack one of the two basic domains that are essential for DNA binding. While the transport of a peptidic agent into cells often poses a severe obstacle to its therapeutic use, we show that a 46-residue leucine zipper peptide can be transported into HeLa cells by coupling it to a 17-residue carrier peptide from the Antennapedia homeodomain, thus paving the way for detailed studies of the therapeutic potential of superzipper peptides.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

Diagnostic performance of Antineutrophil Cytoplasmic Antibody tests for Idiopathic Vasculitides: Metaanalysis with a focus on antimyeloperoxidase antibodies

Objective. The diagnostic value of tests for antimyeloperoxidase antibodies (anti-MPO) for systemic vasculitis is less established than that for cytoplasmic antineutrophil cytoplasmic antibody (cANCA)/antiproteinase 3 antibodies (anti-PR3). Controversy exists regarding the optimal utilization of indirect immunofluorescence (IIF) ANCA testing versus antigen-specific ANCA testing. To summarize the pertinent data, we conducted a metaanalysis examining the diagnostic value of ANCA testing systems that include assays for anti-MPO. Methods. We performed a structured Medline search and reference list review. Target articles in the search strategy were those reporting the diagnostic value of immunoassays for anti-MPO for the spectrum of systemic necrotizing vasculitides that includes Wegener's granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, and isolated pauci-immune necrotizing or crescentic glomerulonephritis, regardless of other types of ANCA tests. Inclusion criteria required specification of a consecutive or random patient selection method and the use of acceptable criteria for the diagnosis of vasculitis exclusive of ANCA test results. Weighted pooled summary estimates of sensitivity and specificity were calculated for anti-MPO alone, anti-MPO + perinuclear ANCA (pANCA), and anti-MPO/pANCA + anti-PR3/cANCA. Results. Of 457 articles reviewed, only 7 met the selection criteria. Summary estimates of sensitivity and specificity (against disease controls only) of assays for anti-MPO for the diagnosis of systemic necrotizing vasculitides were 37.1% (confidence interval 26.6% to 47.6%) and 96.3% (CI 94.1% to 98.5%), respectively. When the pANCA pattern by IIF was combined with anti-MPO testing, the specificity improved to 99.4%, with a lower sensitivity, 31.5%. The combined ANCA testing system (anti-PR3/cANCA + anti-MPO/pANCA) increased the sensitivity to 85.5% with a specificity of 98.6%. Conclusion. These results suggest that while anti-MPO is relatively specific for the diagnosis of systemic vasculitis, the combination system of immunoassays for anti-MPO and IIF for pANCA is highly specific and both tests should be used together given the high diagnostic precision required for these conditions. Because patients with ANCA associated vasculitis have either anti-MPO with pANCA or anti-PR3 with cANCA, and rarely both, a combined ANCA testing system including anti-PR3/cANCA and anti-MPO/pANCA is recommended to optimize the diagnostic performance of ANCA testing. (J Rheumatol 2001;28:1584-90)

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells, In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone, These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.