<p>The plant cyclin-dependent kinases (CDKs) are subjected to the regulation by the interactors/inhibitors of CDK (ICKs). Seven members of the ICK family are known in the plant <i>Arabidopsis thaliana</i>. It has been shown that the N-terminal region of ICK1 makes it unstable in plants, although the mechanism was unknown.</p> <p>In this thesis, to determine role of the N-terminal region in other ICKs, full length ICK2 to ICK7 were compared to truncated mutants lacking the N-terminal region. Results from yeast two-hybrid studies suggest that not all the N-terminal regions in different ICKs have a role similar to the N-terminal region of ICK1. Studies of a set of HA-tagged ICK1 deletion mutants in yeast mapped the critical sequence for ICK1 degradation to the N-terminal 21^st - 40^th amino acid residues. Overexpression of deletion mutants in Arabidopsis also showed that deletion of the 20-amino-acid region of ICK1 lead to a high level of HA-tagged mutant protein, supporting that this region plays a major role in ICK1 degradation <i>in vivo</i>.</p> <p>Treating yeast cells expressing HA-tagged ICK1 with the 26S proteasome inhibitor MG132 moderately increased the level of ICK1 protein, suggesting that 26S proteasome may be involved in the degradation of ICK1. To determine the possible involvement of the two E3 complexes, the Skp1-Cullin-F-box (SCF) complex and anaphase promoting complex/cyclosome (APC/C), a set of yeast mutants defective in either SCF complex or APC/C, were used to express ICK1, ICK1^ÎN20 (ICK1 lacking the N-terminal 20 amino acids) and ICK1^ÎN40. ICK1^ÎN40 showed a very high level of expression in SCF defective mutants, but not in APC/C defective mutants. However, ICK1 and ICK1^ÎN20 did not accumulate to a high level in either of the two types of mutants. These results suggest that two pathways are involved in the degradation of ICK1.</p> <p>Results from this study provide new understanding regarding the role of N-terminal region of ICK1 in conferring protein instability, and the differences among ICKs. They also raise new questions for future investigation on this family of plant cell cycle regulators.</p>

Les canaux ioniques occupent une place primordiale dans les mécanismes du vivant, et leurs dysfonctionnements sont à l'origine de nombreuses pathologies. Dans l'optique d'envisager un traitement efficace de ces maladies, la compréhension des mécanismes de fonctionnement des canaux ioniques constitue un objectif majeur à atteindre. Les toxines animales actives sur les canaux ioniques apparaissent aujourd'hui comme un outil de choix pour mieux comprendre le fonctionnement de ces canaux. Au travers de son introduction bibliographique, ce manuscrit présente les canaux K+, Na+ et Ca2+ et les toxines animales dont ces canaux sont les cibles naturelles. Ces toxines s'organisent majoritairement autour de deux motifs structuraux distincts : le motif CsΑΒ (Cystine stabilized ΑΒ motif), décrit principalement dans les toxines de scorpion actives sur les canaux K+ et Na+, et le motif ICK (Inhibitor Cystine Knot) qui est rencontré dans certaines toxines de cônes et d'araignées actives sur les canaux K+, Na+ ou Ca2+. Le travail effectué au cours de cette thèse est sous-tendu par un objectif, améliorer encore la connaissance et la compréhension de l'interaction d'une toxine et de sa cible, le canal. Les résultats obtenus s'articulent autour de la détermination de la structure RMN de plusieurs toxines. Concernant le motif CsΑΒ, nos résultats montrent que, par conception de protéines chimères, nous pouvons modifier la spécificité et/ou la sélectivité des toxines envers les canaux K+. Concernant le motif ICK, nos résultats pour les toxines actives sur les canaux K+ doivent être confirmés par des études complémentaires puisque la structure indique que ces toxines semblent interagir au niveau du pore du canal, hypothèse en contradiction avec les données biologiques ; pour les toxines actives sur les canaux Ca2+, la spécificité envers les différents types de canaux et l'influence de l'anisotropie de répartition des charges électrostatiques sur l'interaction toxine/canal sont discutées.

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.