The Experts below are selected from a list of 2523 Experts worldwide ranked by ideXlab platform
David J. Craik - One of the best experts on this subject based on the ideXlab platform.
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In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis.
Journal of natural products, 2021Co-Authors: Tien T. Dang, Lai Yue Chan, Benjamin J. Tombling, Peta J. Harvey, Edward K. Gilding, David J. CraikAbstract:Cyclotides are plant-derived peptides that have attracted interest as biocides and scaffolds for the development of stable peptide therapeutics. Cyclotides are characterized by their cyclic backbone and Cystine Knot framework, which engenders them with remarkably high stability. This study reports the Cystine Knot-related peptidome of Rinorea bengalensis, a small rainforest tree in the Violaceae family that is distributed from Australia westward to India. Surprisingly, many more acyclic Knotted peptides (acyclotides) were discovered than cyclic counterparts (cyclotides), with 32 acyclotides and 1 cyclotide sequenced using combined transcriptome and proteomic analyses. Nine acyclotides were isolated and screened against a panel of mammalian cell lines, showing they had the cytotoxic properties normally associated with cyclotide-like peptides. NMR analysis of the acyclotide ribes 21 and 22 and the cyclotide ribe 33 confirmed that these peptides contained the Cystine Knot structural motif. The bracelet-subfamily cyclotide ribe 33 was amenable to chemical synthesis in reasonable yield, an achievement that has long eluded previous attempts to synthetically produce bracelet cyclotides. Accordingly, ribe 33 represents an exciting new bracelet cyclotide scaffold that can be subject to chemical modification for future molecular engineering applications.
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Holocyclotoxin-1, a Cystine Knot toxin from Ixodes holocyclus
Toxicon : official journal of the International Society on Toxinology, 2014Co-Authors: S. Vink, Norelle L Daly, David J. Craik, Natalie Steen, Paul F. AlewoodAbstract:In the past 100 years minimal venom research has focused on ticks despite several species possessing a highly paralytic and lethal venom cocktail of proteinaceous molecules. The saliva of the Australian paralysis tick, Ixodes holocyclus, has been responsible for 20 human fatalities from 1900 to 1945, and up to 100,000 domestic animal fatalities annually. In the last 50 years, research on this tick has focused on identifying the neurotoxins present in the saliva and in the last ten years the sequence of a potential neurotoxin, HT-1, has been determined. In this study we chemically synthesised HT-1 using Boc-chemistry in combination with native chemical ligation. Following successful oxidative folding, we determined the three-dimensional structure of HT-1 by NMR spectroscopy and found a novel structural fold with three of the four disulfide bonds comprising the inhibitory Cystine Knot (ICK) motif. The fourth disulfide bond connects the second loop to the N-terminal, which decreases the flexibility of the structure.
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A comparative study of extraction methods reveals preferred solvents for Cystine Knot peptide isolation from Momordica cochinchinensis seeds
Fitoterapia, 2014Co-Authors: Tunjung Mahatmanto, Aaron G. Poth, Joshua S. Mylne, David J. CraikAbstract:MCoTI-I and MCoTI-II (short for Momordica cochinchinensis Trypsin Inhibitor-I and -II, respectively) are attractive candidates for developing novel intracellular-targeting drugs because both are exceptionally stable and can internalize into cells. These seed-derived Cystine Knot peptides are examples of how natural product discovery efforts can lead to biomedical applications. However, discovery efforts are sometimes hampered by the limited availability of seed materials, highlighting the need for efficient extraction methods. In this study, we assessed five extraction methods using M. cochinchinensis seeds, a source of well-characterized Cystine Knot peptides. The most efficient extraction of nine known Cystine Knot peptides was achieved by a method based on acetonitrile/water/formic acid (25:24:1), followed by methods based on sodium acetate (20 mM, pH 5.0), ammonium bicarbonate (5 mM, pH 8.0), and boiling water. On average, the yields obtained by these four methods were more than 250-fold higher than that obtained using dichloromethane/methanol (1:1) extraction, a previously applied standard method. Extraction using acetonitrile/water/formic acid (25:24:1) yielded the highest number of reconstructed masses within the majority of plant-derived Cystine Knot peptide mass range but only accounted for around 50% of the total number of masses, indicating that any single method may result in under-sampling. Applying acetonitrile/water/formic acid (25:24:1), boiling water, and ammonium bicarbonate (5 mM, pH 8.0) extractions either successively or discretely significantly increased the sampling number. Overall, acetonitrile/water/formic acid (25:24:1) can facilitate efficient extraction of Cystine-Knot peptides from M. cochinchinensis seeds but for discovery purposes the use of a combination of extraction methods is recommended where practical.
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Novel Inhibitor Cystine Knot Peptides from Momordica charantia
PloS one, 2013Co-Authors: Lai Yue Chan, Richard J Clark, Norelle L Daly, David J. Craik, Jun Tang, Guang-zhi Zeng, Octavio L. Franco, Cinzia Cantacessi, Ning-hua TanAbstract:Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor Cystine Knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.
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A new family of Cystine Knot peptides from the seeds of Momordica cochinchinensis
Peptides, 2012Co-Authors: Lai Yue Chan, David J. Craik, Guang-zhi Zeng, Ning-hua Tan, Norelle L DalyAbstract:Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor Cystine Knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3-MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK( peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active. (C) 2012 Elsevier Inc. All rights reserved.
Maria A Schumacher - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of human type iii collagen gly991 gly1032 Cystine Knot containing peptide shows both 7 2 and 10 3 triple helical symmetries
Journal of Biological Chemistry, 2008Co-Authors: Sergei P Boudko, Jurgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter Bachinger, Maria A SchumacherAbstract:Abstract Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal Cystine Knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal Cystine Knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly991–Gly1032 structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the Cystine Knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.
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Crystal Structure of Human Type III Collagen Gly991–Gly1032 Cystine Knot-containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries
The Journal of biological chemistry, 2008Co-Authors: Sergei P Boudko, Jurgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter Bachinger, Maria A SchumacherAbstract:Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal Cystine Knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)(n) or (Gly-Pro-Hyp)(n). To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal Cystine Knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly(991)-Gly(1032) structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the Cystine Knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.
Walter David Obregón - One of the best experts on this subject based on the ideXlab platform.
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Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.
International journal of molecular sciences, 2018Co-Authors: Juliana Cotabarren, Mariana Edith Tellechea, Sebastian Tanco, Julia Lorenzo, Javier Garcia-pardo, Francesc X. Avilés, Walter David ObregónAbstract:Cystine-Knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic Knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular Knotted structure. The Knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel Cystine-Knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churquena. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.
Harald Kolmar - One of the best experts on this subject based on the ideXlab platform.
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Cystine Knot peptides targeting cancer relevant human cytotoxic t lymphocyte associated antigen 4 ctla 4
Journal of Peptide Science, 2015Co-Authors: Franziska Maas, Hans-ulrich Schmoldt, Olga Avrutina, Michael Reinwarth, Matin Daneschdar, Ugur Sahin, Bernhard Valldorf, Joycelyn Wustehubelausch, Stephan Dickgieser, Harald KolmarAbstract: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.
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Cystine‐Knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)
Journal of peptide science : an official publication of the European Peptide Society, 2015Co-Authors: Franziska Maaß, Hans-ulrich Schmoldt, Olga Avrutina, Michael Reinwarth, Joycelyn Wüstehube-lausch, Matin Daneschdar, Ugur Sahin, Stephan Dickgießer, Bernhard Valldorf, Harald KolmarAbstract: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.
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Fragmentation Follows Structure: Top-Down Mass Spectrometry Elucidates the Topology of Engineered Cystine-Knot Miniproteins
PloS one, 2014Co-Authors: Michael Reinwarth, Olga Avrutina, Sebastian Fabritz, Harald KolmarAbstract:Over the last decades the field of pharmaceutically relevant peptides has enormously expanded. Among them, several peptide families exist that contain three or more disulfide bonds. In this context, elucidation of the disulfide patterns is extremely important as these motifs are often prerequisites for folding, stability, and activity. An example of this structure-determining pattern is a Cystine Knot which comprises three constrained disulfide bonds and represents a core element in a vast number of mechanically interlocked peptidic structures possessing different biological activities. Herein, we present our studies on disulfide pattern determination and structure elucidation of Cystine-Knot miniproteins derived from Momordica cochinchinensis peptide MCoTI-II, which act as potent inhibitors of human matriptase-1. A top-down mass spectrometric analysis of the oxidised and bioactive peptides is described. Following the detailed sequencing of the peptide backbone, interpretation of the MS3 spectra allowed for the verification of the Knotted topology of the examined miniproteins. Moreover, we found that the fragmentation pattern depends on the Knottin’s folding state, hence, tertiary structure, which to our knowledge has not been described for a top-down MS approach before.
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combinatorial optimization of Cystine Knot peptides towards high affinity inhibitors of human matriptase 1
PLOS ONE, 2013Co-Authors: Bernhard Glotzbach, Olga Avrutina, Michael Reinwarth, Michael Tomaszowski, Sebastian Fabritz, Niklas Weber, Heiko Fittler, Andreas Christmann, Harald KolmarAbstract:Cystine-Knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain Cystine-Knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of Cystine-Knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.
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Oxidative folding of peptides with Cystine-Knot architectures: kinetic studies and optimization of folding conditions.
Chembiochem : a European journal of chemical biology, 2012Co-Authors: Michael Reinwarth, Olga Avrutina, Bernhard Glotzbach, Michael Tomaszowski, Sebastian Fabritz, Harald KolmarAbstract:Bioactive peptides often contain several disulfide bonds that provide the main contribution to conformational rigidity and structural, thermal, or biological stability. Among them, Cystine-Knot peptides-commonly named "Knottins"-make up a subclass with several thousand natural members. Hence, they are considered promising frameworks for peptide-based pharmaceuticals. Although Cystine-Knot peptides are available through chemical and recombinant synthetic routes, oxidative folding to afford the bioactive isomers still remains a crucial step. We therefore investigated the oxidative folding of ten protease-inhibiting peptides from two Knottin families, as well as that of an HIV entry inhibitor and of aprotinin, under two conventional sets of folding conditions and by a newly developed procedure. Kinetic studies identified folding conditions that resulted in correctly folded miniproteins with high rates of conversion even for highly hydrophobic and aggregation-prone peptides in concentrated solutions.
Sergei P Boudko - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of human type iii collagen gly991 gly1032 Cystine Knot containing peptide shows both 7 2 and 10 3 triple helical symmetries
Journal of Biological Chemistry, 2008Co-Authors: Sergei P Boudko, Jurgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter Bachinger, Maria A SchumacherAbstract:Abstract Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal Cystine Knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal Cystine Knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly991–Gly1032 structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the Cystine Knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.
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Crystal Structure of Human Type III Collagen Gly991–Gly1032 Cystine Knot-containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries
The Journal of biological chemistry, 2008Co-Authors: Sergei P Boudko, Jurgen Engel, Kenji Okuyama, Kazunori Mizuno, Hans Peter Bachinger, Maria A SchumacherAbstract:Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal Cystine Knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)(n) or (Gly-Pro-Hyp)(n). To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal Cystine Knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly(991)-Gly(1032) structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the Cystine Knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.
