| 1. |
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| 2. |
- Babbitt, Patricia C., et al.
(författare)
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Creating a specialist protein resource network : a meeting report for the protein bioinformatics and community resources retreat
- 2015
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Ingår i: Database. - : Oxford University Press (OUP). - 1758-0463.
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Tidskriftsartikel (refereegranskat)abstract
- During 11-12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication and funding. An important outcome of this meeting was the creation of a Specialist Protein Resource Network that we believe will improve coordination of the activities of its member resources. We invite further protein database resources to join the network and continue the dialogue.
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| 3. |
- Koehbach, Johannes, et al.
(författare)
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Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:52, s. 21183-21188
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V-1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.
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| 4. |
- Aboye, Teshome Leta, et al.
(författare)
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Interlocking disulfides in circular proteins : toward efficient oxidative folding of cyclotides.
- 2011
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Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert Inc. - 1523-0864 .- 1557-7716. ; 14:1, s. 77-86
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are ultrastable plant proteins characterized by the presence of a cyclic amide backbone and three disulfide bonds that form a cystine knot. Because of their extreme stability, there has been significant interest in developing these molecules as a drug design scaffold. For this potential to be realized, efficient methods for the synthesis and oxidative folding of cyclotides need to be developed, yet we currently have only a basic understanding of the folding mechanism and the factors influencing this process. In this study, we determine the major factors influencing oxidative folding of the different subfamilies of cyclotides. The folding of all the cyclotides examined was heavily influenced by the concentration of redox reagents, with the folding rate and final yield of the native isomer greatly enhanced by high concentrations of oxidized glutathione. Addition of hydrophobic solvents to the buffer also enhanced the folding rates and appeared to alter the folding pathway. Significant deamidation and isoaspartate formation were seen when oxidation conditions were conducive to slow folding. The identification of factors that influence the folding and degradation pathways of cyclotides will facilitate the development of folding screens and optimized conditions for producing cyclotides and grafted analogs as stable peptide-based therapeutics.
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| 5. |
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| 6. |
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| 7. |
- Andersson, Håkan S., et al.
(författare)
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The alpha-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance
- 2012
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Ingår i: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 43:4, s. 1471-1483
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Tidskriftsartikel (refereegranskat)abstract
- Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the alpha-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg(7) or Glu(15) residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg(7) and Glu(15) substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic alpha-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical alpha-defensin salt-bridge facilitates adoption of the characteristic alpha-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.
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| 8. |
- Burman, Robert, 1979-, et al.
(författare)
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Evaluation of toxicity and anti-tumour activity of cycloviolacin O2 in mice.
- 2010
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 94:5, s. 626-634
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Tidskriftsartikel (refereegranskat)abstract
- Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v. 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v. 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC50 values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2–3 days; lower doses were without complications.
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| 9. |
- Craik, David J., et al.
(författare)
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Nomenclature of homodetic cyclic peptides produced from ribosomal precursors : An IUPAC task group interim report
- 2016
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 106:6, s. 917-924
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Tidskriftsartikel (refereegranskat)abstract
- In 2015, an International Union of Pure and Applied Chemistry (IUPAC) Task Group was formed to develop nomenclature recommendations for homodetic cyclic peptides produced from ribosomal precursors. Delegates of the 2015 International Conference on Circular Proteins (ICCP) were presented with the strengths and weaknesses of four published approaches to homodetic cyclic peptide nomenclature, and a summary of the ensuing discussion is presented here. This interim report presents a potentially novel suggestion-the use of Cahn-Ingold-Prelog rules to specify amino acid priority in homodetic peptides for consistent numbering. Indeed, this might be the first extension of the Cahn-Ingold-Prelog rules in five decades. The authors invite interested parties to contact the corresponding author with suggestions for the improvement of the proposed nomenclature; these ideas will be discussed and considered for inclusion in the final report.
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| 10. |
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| 11. |
- de Veer, Simon J., et al.
(författare)
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Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition
- 2015
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 469:2, s. 243-253
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Tidskriftsartikel (refereegranskat)abstract
- Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.
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| 12. |
- Gruber, Christian W, et al.
(författare)
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Distribution and evolution of circular miniproteins in flowering plants
- 2008
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Ingår i: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 20:9, s. 2471-2483
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids ( approximately 125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.
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| 13. |
- Leta Aboye, Teshome, et al.
(författare)
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Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2
- 2008
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 9:1, s. 103-113
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Tidskriftsartikel (refereegranskat)abstract
- The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.
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| 14. |
- Nilsson, K Peter R, et al.
(författare)
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Solution structure of chi-conopeptide MrIA, a modulator of the human norepinephrine transporter.
- 2005
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 80:6, s. 815-823
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Tidskriftsartikel (refereegranskat)abstract
- The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.
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| 15. |
- Plan, Manuel Rey R, et al.
(författare)
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The cyclotide fingerprint in Oldenlandia affinis : elucidation of chemically modified, linear and novel macrocyclic peptides
- 2007
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:9, s. 1001-1011
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Tidskriftsartikel (refereegranskat)abstract
- The complete suite of cyclotides present in Oldenlandia affinis (Rubiaceae), the plant that was originally found to contain this unique family of circular proteins, has been characterised. This study expands the number of known cyclotides in this plant to 17, of which nine new sequences (kalata B9-B17) were characterised in this work. In addition, five derivatives that contain oxidation products of the conserved tryptophan were identified, and it was shown that the formation of these derivatives is catalysed by exposure to sunlight. Furthermore, we describe two linear cyclotide analogues. These acyclic peptides have three intact disulfide bonds, and their N and C termini coincide with the hypothesised cleavage sites from the precursor protein. This work increases our knowledge about the sequence variation that is accommodated by the cyclic cystine knot scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for cyclotides. These pathways have important implications for the persistence and environmental fate of the cyclotides if used as crop-protection agents.
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| 16. |
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| 17. |
- Simonsen, Shane M, et al.
(författare)
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A continent of plant defense peptide diversity : cyclotides in Australian Hybanthus (Violaceae)
- 2005
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Ingår i: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 17:11, s. 3176-3189
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.
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| 18. |
- Wang, Conan K L, et al.
(författare)
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Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis
- 2008
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Ingår i: Journal of natural products (Print). - : American Chemical Society (ACS). - 0163-3864 .- 1520-6025. ; 71:1, s. 47-52
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are macrocyclic plant peptides characterized by a knotted arrangement of three disulfide bonds. They display a range of interesting bioactivities, including anti-HIV and insecticidal activities. More than 100 different cyclotides have been isolated from two phylogenetically distant plant families, the Rubiaceae and Violaceae. In this study we have characterized the cyclotides from Viola yedoensis, an important Chinese herb from the Violaceae family that has been reported to contain potential anti-HIV agents. From V. yedoensis five new and three known cyclotides were identified and shown to have anti-HIV activity. The most active of these is cycloviolacin Y5, which is one of the most potent of all cyclotides tested so far using in vitro XTT-based anti-HIV assays. Cycloviolacin Y5 is the most hydrophobic of the cyclotides from V. yedoensis. We show that there is a positive correlation between the hydrophobicity and the anti-HIV activity of the new cyclotides and that this trend tracks with their ability to disrupt membranes, as judged from hemolytic assays on human erythrocytes.
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| 19. |
- Burman, Robert, et al.
(författare)
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Cyclotide proteins and precursors from the genus Gloeospermum : filling a blank spot in the cyclotide map of Violaceae
- 2010
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Ingår i: Phytochemistry. - : Elsevier BV. - 0031-9422 .- 1873-3700. ; 71:1, s. 13-20
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.
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| 20. |
- Burman, Robert, et al.
(författare)
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Distribution of circular proteins in plants : large-scale mapping of cyclotides in the Violaceae
- 2015
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- During the last decade there has been increasing interest in small circular proteins found in plants of the violet family (Violaceae). These so-called cyclotides consist of a circular chain of approximately 30 amino acids, including six cysteines forming three disulfide bonds, arranged in a cyclic cystine knot (CCK) motif. In this study we map the occurrence and distribution of cyclotides throughout the Violaceae. Plant material was obtained from herbarium sheets containing samples up to 200 years of age. Even the oldest specimens contained cyclotides in the preserved leaves, with no degradation products observable, confirming their place as one of the most stable proteins in nature. Over 200 samples covering 17 of the 23-31 genera in Violaceae were analyzed, and cyclotides were positively identified in 150 species. Each species contained a unique set of between one and 25 cyclotides, with many exclusive to individual plant species. We estimate the number of different cyclotides in the Violaceae to be 5000-25,000, and propose that cyclotides are ubiquitous among all Violaceae species. Twelve new cyclotides from six phylogenetically dispersed genera were sequenced. Furthermore, the first glycosylated derivatives of cyclotides were identified and characterized, further increasing the diversity and complexity of this unique protein family.
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| 21. |
- Gerlach, Samantha L., et al.
(författare)
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A Systematic Approach to Document Cyclotide Distribution in Plant Species from Genomic, Transcriptomic, and Peptidomic Analysis
- 2013
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 100:5, s. 433-437
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are a large family of plant peptides characterized by their cyclic cystine knot composed of a circular backbone and three disulfide bonds that impart exceptional stability. They, and several acyclic variants, have been isolated from plants within the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families. A variety of chemical and genetic approaches have been applied for the discovery and characterization of cyclotides. As investigations of cyclotide expression, distribution, and phylogeny rapidly increase, the authors have proposed the inclusion of information pertaining to plant species that have been analyzed but do not appear to express cyclotides into the CyBase database. CyBase is dedicated to providing web tools and information about cyclic peptides and proteins to the scientific community. Including detailed information about sampling and analysis parameters of plant species that have been investigated but not published elsewhere should assist in the process of selecting species for establishing new cyclotide discovery projects, as well as for detailed reanalysis using alternative technical approaches. In summary, the collection and deposition of all plant species that have been examined (whether cyclotides have been found or not) would help to impart a deeper understanding of cyclotide discovery, evolution, and physiological function.
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| 22. |
- Göransson, Ulf, et al.
(författare)
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Disulfide mapping of the cyclotide kalata B1 : Chemical proof of the cystic cystine knot motif
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:48, s. 48188-48196
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Tidskriftsartikel (refereegranskat)abstract
- The cyclotides are a recently discovered family of plant proteins that have the fascinating structural feature of a continuous cyclic backbone and, putatively, a knotted arrangement of their three conserved disulfide bonds. We here show definite chemical proof of the I-IV, II-V, III-VI knotted disulfide connectivity of the prototypic cyclotide kalata B1. This has been achieved by a new approach for disulfide analysis, involving partial reduction and stepwise alkylation including introduction of charges and enzymatic cleavage sites by aminoethylation of cysteines. The approach overcomes the intrinsic difficulties for disulfide mapping of cyclotides, i.e. the cyclic amide backbone, lack of cleavage sites between cysteines, and a low or clustered content of basic amino acids, and allowed a direct determination of the disulfide bonds in kalata B1 using analysis by mass spectrometry. The established disulfide connectivity is unequivocally shown to be cystine knotted by a topological analysis. This is the first direct chemical determination of disulfides in native cyclotides and unambiguously confirms the unique cyclic cystine knot motif.
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| 23. |
- Haugaard-Kedström, Linda M., et al.
(författare)
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Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin-2.
- 2015
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:3, s. 891-900
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Tidskriftsartikel (refereegranskat)abstract
- Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.
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| 24. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
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Structural Properties of Relaxin Chimeras: NMR Characterization of the R3/I5 Relaxin Peptide
- 2009
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1160, s. 27-30
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Tidskriftsartikel (refereegranskat)abstract
- Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor-ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure-activity data on relaxins.
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| 25. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
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Structure of the human insulin-like peptide 5 and characterization of conserved hydrogen bonds and electrostatic interactions within the relaxin framework
- 2009
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 419, s. 619-627
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Tidskriftsartikel (refereegranskat)abstract
- INSL5 (insulin-like peptide 5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and, although the fulfil biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon, as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signalling. INSL5 activates the relaxin family peptide receptor 4 with high potency and appears to be the endogenous ligand for this receptor, on the basis of overlapping expression profiles and their apparent co-evolution. In the present Study, we have used solution-state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three helical segments that are braced by three disulfide bonds and enclose a hydrophobic core. Furthermore, we characterized in detail the hydrogen-bond network and electrostatic interactions between charged groups in INSL5 by NMR-monitored temperature and pH titrations and Undertook a comprehensive structural comparison with other members of the relaxin family, thus identifying the conserved structural features of the relaxin fold. The B-chain helix, which is the primary receptor-binding site of the relaxins, is longer in INSL5 than in its close relative relaxin-3. As this feature results in a different positioning of the receptor-activation domain Arg(B23) and Trp(B24), it may be an important contributor to the difference in biological activity observed for these two peptides. Overall, the structural Studies provide mechanistic insights into the receptor selectivity of this important family of hormones.
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