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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Cyclotides evolve : Studies on their natural distribution, structural diversity, and activity

Park, Sungkyu January 2016 (has links)
The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus. In addition, the structural diversity of the cyclotides was related to specific features of the sequences of their precursor proteins, their evolutionary selection and expression levels. A number of studies have suggested that the biological activities of the cyclotides are due to their ability to interact with and disrupt biological membranes. To better explain this behavior, quantitative structure-activity relationship (QSAR) models were developed to link the cyclotides’ biological activities to the membrane-interactive physicochemical properties of their molecular surfaces. Both scalar quantities (such as molecular surface areas) and moments (such as the distributions of specific properties over the molecular surface) were systematically taken into account in the development of these models. This approach allows the physicochemical properties of cyclotides to be geometrically interpreted, facilitating the development of guidelines for drug design using cyclotide scaffolds. Finally, an optimized microwave-assisted Fmoc-SPSS procedure for the total synthesis of cyclotides was developed. Microwave irradiation is used to accelerate and improve all the key steps in cyclotide synthesis, including the assembly of the peptide backbone by Fmoc-SPPS, the cleavage of the protected peptide, and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation.
2

Engineering of the Ultra-stable Cystine Knot Framework of Microproteins : Design, Chemical Synthesis and Structural Studies

Aboye, Teshome Leta January 2011 (has links)
Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations. Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used. Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.
3

Diversité structurale et d'activité biologique des Albumines entomotoxiques de type 1b des graines de Légumineuses

Louis, Sandrine 20 February 2004 (has links) (PDF)
PA1b (Pea Albumin 1 sous-unité b), une knottine toxique de 37 acides aminés, présente un grand intérêt dans la lutte contre les charançons des céréales (Sitophilus spp.), principaux ravageurs des céréales stockées.<br />Afin de mieux connaître la nouvelle famille peptidique de PA1b, sa variabilité tant structurale que d'activité biologique a été étudiée au sein des Légumineuses. Après avoir validé notre approche sur 4 espèces végétales "test", nous avons caractérisé 24 gènes homologues chez 18 espèces de Papilionoideae. De plus, l'activité insecticide d'extraits de graines de 60 espèces des trois sous-familles de Légumineuses a été déterminée sur charançons de souche sensible et résistante à PA1b. Afin de relier variations de structure et d'activité, une approche par mutagenèse dirigée a été envisagée. Un système d'expression bactérienne et de purification de PA1b a été mis au point. Bien que de masse conforme (cystéines oxydées), le peptide recombinant ne présente pas d'activité biologique.
4

Cytotoxic Cyclotides : Structure, Activity, and Mode of Action

Svangård, Erika January 2005 (has links)
Cyclotides are small cyclic plant proteins, and this thesis addresses their cytotoxic structure-activity properties and their mode of action on human cancer cell lines. Cyclotides were isolated from Viola odorata and Viola tricolor; three novel cyclotide sequences and two known sequences, but of new origin, were identified using mass spectrometry, amino acid analysis, and Edman degradation. The cyclotide structure includes three disulphide bonds in a knotted arrangement, which forces hydrophobic amino acid residues to be exposed on the surface of the molecule; 3-D homology models of cyclotides have revealed an amphipathic surface and charged residues located at similar positions in the molecules. The charged amino acid residues were shown to play a key role in the cytotoxicity of the cyclotide cycloviolacinO2 on a human lymphoma cell line. Methylation of Glu caused a dramatic change in cytotoxicity, lowering the potency 48 times, whereas concealing the charge of Arg with 1,2-cyclohexanedione caused virtually no change in potency. Acetylation of the two Lys caused a 3-fold reduction in potency, and masking all positive charges caused a 7-fold reduction. Additionally, disturbing the amphipathic structure by reducing and alkylating the disulphide bonds abolished the cytotoxicity. The time dependency of cytotoxicity and cell gross morphology after cyclotide exposure were investigated on the lymphoma cell line. Cells exposed to 4 µM of cycloviolacinO2 showed necrotic characteristics, such as membrane disintegration, within 5 min; a membrane disruptive effect of cycloviolacinO2 was also observed in a functional assay based on liposomes at a peptide-to-lipid molar ratio of 6.5. The anti-tumour properties of cycloviolacinO2 were evaluated on three human cancer cell lines using the hollow fibre assay in vitro and in vivo. The cyclotide exhibited potent anti-tumour activity in the micro-molar concentration range on all cell lines in vitro, but no effect on tumour growth could be established in vivo.
5

Structure and Activity of Circular Plant Proteins : Cytotoxic Effects of Viola Cyclotides

Herrmann, Anders January 2007 (has links)
Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency
6

Contributions To Venominformatics : Sequence-Structure-Function Studies Of Toxins From Marine Cone Snails. Application Of Order-Statistics Filters For Detecting Membrane-Spanning Helices

Mondal, Sukanta 02 1900 (has links)
Venomous animals have evolved a vast array of peptide toxins for prey capture and defense. Nature has evolved the venoms into a huge library of active molecules with high selectivity and affinity, which could be explored as therapeutics or serve as a template for drug design. The individual components of venom i.e. toxins are used in ion channel and receptor studies, drug discovery, and formulation of insecticides. ‘Venominformatics is a systematic bioinformatics approach in which classified, consolidated and cleaned venom data are stored into repositories and integrated with advanced bioinformatics tools and computational biology for the analysis of structure and function of toxins.’ Conus peptides (conopeptides), the main components of Conus venom, represent a unique arsenal of neuropharmacologically active molecules that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes and neuronal receptors. Ziconotide (ω-conotoxin MVIIa from Conus magus (Magician's cone snail)), is proven as an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain (U.S. Food and Drug Administration. Center for Drug Evaluation and Research) attesting to the pharmaceutical importance of Conus peptides. From the point of view of protein sequence and structure analysis, conopeptides can serve as attractive systems for the studies in sequence comparison, pattern extraction, structure–function correlations, protein–protein interactions and evolutionary analysis. Despite their importance and extensive experimental investigations on them, they have been hardly explored through in silico methods. The present thesis is perhaps the first attempt at deploying a multi-pronged bioinformatics approaches for studies in the burgeoning field of conopeptides. In the process of sequence-structure-function studies of conopeptides, we have created several sequence patterns of different conopeptide families and these have been accepted for inclusion in international databases such as PROSITE, the first pattern database to have been developed (http://www.expasy.org/prosite) and INTERPRO (http://www.ebi.ac.uk/interpro). More importantly, we have carried out extensive literature survey on the peptides for which we have defined the patterns to create PROSITE compatible documentation files (PDOC6004, PDOC60025 and PDOC60027). We have also created a series of sequence patterns and associated documentation filesof pharmaceutically promising peptides from plants and venomous animals (including O-conotoxin and P-conotoxin superfamily members) with knottin scaffold. Knottins provide appealing scaffolds for protein engineering and drug design due to their small size, high structural stability, strong sequence tolerance and easy access to chemical synthesis. The sequence patterns and associated documentation files created by us should be useful in protein family classification and functional annotation. Even though patterns might be useful at the family level, they may not always be adequate at the superfamily level due to hypervariability of mature toxins. In order to overcome this problem, we have demonstrated the applicationos of multi-class support vector machines (MC-SVMs) for the successful in silico classification of the mature conotoxins into their superfamilies. TheI- and J-conotoxin-superfamily members were analyzed in greater detail. On the basis of in silico analysis, we have divided the 28 entries previously grouped as I-conotoxin superfamily in UniProtKB/Swiss-Prot (release 49.0) into I1 and I2 superfamilies inview of their having two different types of signal peptides and exhibiting distinct functions. A comparative study of the theoretically modeled structure of ViTx from Conus virgo, a typical member of I2-conotoxin superfamily, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels. Putative complexes created by us of very recently characterized J-superfamily conotoxin p11-4a with Kv1.6 suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. This finding provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profile against subtypes of the potassium channels for these conopeptides. Furthermore, the present thesis reports the application of order-statistic filters and hydrophobicity profiles for predicting the location of membrane-spanning helices. The Proposed method is in particular effective for the class of helical membrane proteins, namely the therapeutically important voltage-gated ion channels, which are natural targets of several conotoxins. Our suggested ab initio approach is comparatively better than other spatial filters, confirming to the efficacy of including the concept of order or ranking information for prediction of TM helicdes. Such approaches should be of value for improved prediction performance including in large-scale applications. In addition, anlaysis has been carried out of the role of context in the relationship between form and function for the true PDB hits of some nonCys-rich PROSITE patterns. We have found specific examples of true hits of some PROSITE patterns displaying structural plasticity by assuming significantly different local conformation, depending upon the context. The work was carried out as a part of the research interest in our group in studying structural and other features of protein sequence patterns. The Contributions of the candidate to venominormatics include, creation of protein sequence patterns and information highlighting the importance of the patterns as gleaned from the lteratures for family classification: profile HMM and MC-SVMs for conotoxin superfamily classification; in silico characterization of I1 and I2 conotoxin superfamilies; studies of interaction with Kv1 channels of typical members of I2 and 3 conotoxin superfamilies and development of improved methods for detecting membrane-spanning helices. Chapter I starts with a brief account of venominformatics; bioinformatics for venoms and toxins. Chapter 2 presents a regular expression based classification of Conus peptides. Chapter 3 revisits the 28 entries previously grouped as I-conotoxin superfamily in UniProt Swiss-Prot knowledgebase (release 49.0) having four disulfide bonds with Cys arrangement C-C-CC-CC-C-C and they inhibit or modify ion channels of nerve cells. Chapter 4 describes pseudo-amino acid composition and MC-SVMs approach for conotoxin superfamily classification. Chapter 5 describes in silico detection of binding mode with Kv1.6 channel of J-superfamily conotoxin p114a from bermivorouos cone snail, Conus planorbis. Chapter 6 presents a comparative sequence-structure-function analysis of naturally occurring Cys-rich peptides having the Knottin or inhibitor cystine knot(ICK) scaffold, from different plants and venomous animals based on information available in the knottin database(http://knottin.cbs.cnrs.fr/). Chapter 7 describes the application of order-statistic filters and hydrophobicity profiles for detecting membrane-spanning helices. Chapter 8 describes the role of context in the relationship between form and function for the true PDB hits of some non Cys-rich PROSITE patterns. Chapter 9 summaries the important findings of the present studies on naturally occurring bioactive Cys-rich peptides with emphasis on Conus peptides and their interactions with respective target such as voltage-gated ion channels.
7

Neue Enzyminhibitoren und Rezeptoragonisten durch Variation funktionaler Schleifen von Mikroproteinen / New enzyme inhibitors and receptor agonists by variation of functional loops of microproteins

Schmoldt, Hans-Ulrich 28 April 2005 (has links)
No description available.
8

Strukturelle Untersuchungen an Varianten des Ecballium elaterium Trypsin Inhibitors-II (EETI-II) / Structural characterization of variants of the Ecballium elaterium trypsin inhibitor EETI-II

Krätzner, Ralph 27 June 2001 (has links)
No description available.
9

Structural studies of Norrin dependent Wnt/beta-catenin signaling

Chang, Tao-Hsin January 2014 (has links)
Norrin is a secreted cystine-knot growth factor that plays critical roles in vascular development in the brain, retina, and cochlea, as well as the uterus. Although Norrin is unrelated to the lipid-modified morphogens Wnts, Norrin activates the canonical Wnt/&beta;-catenin pathway by binding to receptor Frizzled4 cysteine-rich domain (Fz4-CRD) and co-receptors of low density lipoprotein receptor related protein 5/6 ectodomain (Lrp5/6-ECD) in conjunction with Tetraspanin-12 (Tspan-12). Like Wnts, Norrin has limited extracellular diffusion properties as a result of associating with heparan sulfate proteoglycans (HSPGs). Mutations lead to inherited disordered retinal vascularization diseases such as Norrie disease, familial exudative vitreoretinopathy and coats' disease. However, the molecular mechanism of how Norrin initiates signalling by engagement with Fz4, Lrp5/6, and HSPGs has remained unresolved. Here, novel strategies for protein production of recombinant human Norrin and Fz4-CRD as well as the complex are developed. The crystal structures of Norrin and its complex with Fz4-CRD, plus complex bound with the heparin mimic sucrose octasulphate, and unliganded structures of Fz4-CRD are presented. These structural data together with biophysical and cellular assays not only reveal the Fz4 and Lrp5/6 binding sites on distinct patches of the Norrin surface, but also indicate the HSPGs binding site on Norrin and Fz4-CRD as well as providing a framework to explain numerous disease-related mutations. Structural comparison with Xenopus Wnt8 in complex with mouse Fz8-CRD provides molecular insights for our understanding of ligand-receptor binding specificity and promiscuity, which has important implications for developing therapeutic strategies against Norrin dependent retinal disorders, and cancers caused by abnormal Wnt signaling.

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