Dissertationen zum Thema „PDZ domain proteins“

Abstract The Z-disk is a sophisticated structure that connects adjacent sarcomeres in striated muscle myofibrils. α-Actinin provides strength to the Z-disks by crosslinking the actin filaments of adjacent sarcomeres. α-Actinin is an antiparallel homodimer, composed of an N-terminal actin binding domain (ABD), the central rod domain, and two pairs of C-terminal EF-hands. The PDZ-LIM domain proteins interact with α-actinin at the Z-disk. Of these proteins, only the actinin-associated LIM protein (ALP), Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) and C-terminal LIM protein (CLP36) have a ZASP/Cypher-like (ZM) motif consisting of 26-27 conserved residues in the internal region between the PDZ and LIM domains. The aim of this work was to understand the molecular interplay between the ZM-motif containing members of the PDZ-LIM proteins and α-actinin. To unveil the biological relevance of the interaction between the PDZ-LIM proteins and α-actinin, naturally occurring human ZASP/Cypher mutations were analyzed. Two interaction sites were found between ALP, CLP36 and α-actinin using recombinant purified proteins in surface plasmon resonance (SPR) analysis. The PDZ domain of ALP and CLP36 recognized the C-terminus of α-actinin, whereas the internal regions bound to the rod domain. Further characterization showed that the ALP internal region adopts and extended conformation when interacting with α-actinin and that the ZM-motif partly mediated the interaction, but did not define the entire interaction area. ZASP/Cypher also interacted and competed with ALP in binding to the rod domain. The internal fragments containing the ZM-motif were important for co-localization of ALP and ZASP/Cypher with α-actinin at the Z-disks and on stress fibers. The absence of ALP and ZASP/Cypher in focal contacts indicates that other interacting molecules, for instance vinculin and integrin, may compete in binding to the rod in these areas or additional proteins are required in targeting to these locations. The co-localization of the ZASP/Cypher with α-actinin could be released by disrupting the stress fibers leading to an accumulation of α-actinin in the cell periphery, whereas ZASP/Cypher was not in these areas. This suggests that an intact cytoskeleton is important for ZASP/Cypher interaction with α-actinin. Earlier studies have shown that mutations in the ZASP/Cypher internal region are associated with muscular diseases. These mutations, however, did not affect ZASP/Cypher co-localization with α-actinin or the stability of ZASP/Cypher proteins. The Z-disk possesses a stretch sensor, which is involved in triggering hypertrophic growth as a compensatory mechanism to increased workloads. α-Actinin is a docking site of molecules that are involved in hypertrophic signaling cascades mediated by calsarcin-calcineurin and protein kinase C (PKC) isoforms. The internal interaction site may be involved in targeting PKCs, which bind to the LIM domains of ZASP/Cypher, to the Z-disks. The similar location of the internal interaction site with calsarcin on the rod suggests that ZASP/Cypher, ALP and CLP36 may regulate calsarcin-mediated hypertrophic signaling.

Wnt signalling is one the most important pathways involved in embryonic development. It controls a number of processes including cellular proliferation, stem cells maintenance, cell fate decisions and establishment of tissue polarity. It is also frequently deregulated in human cancers. Frizzled-7 is a member of the Frizzled family responsible for the signal transduction in Wnt signalling. Frizzled-7 has been reported to be upregulated in several types: of cancer. Furthermore, recent reports suggest that endocytosis of Frizzled may play a critical enhancing role in Wnt signal transduction, thus facilitating cancer development. We demonstrate here that the C-terminal PDZ binding motif (PDZ-BM) of Frizzled-7 contributes to signalling triggered by the receptor. We also explore the interaction between Frizzled-7 and syntenin-1, a PDZ domain containing protein that controls endocytic trafficking of various transmembrane proteins. We demonstrate that syntenin-1 regulates Frizzled-7 cell distribution and also modulates canonical Wnt signalling in epithelial breast cancer cells. Further, we report that the C-terminal PDZ-BM of Fz7 is indispensable for the receptor interaction with a number of PDZ proteins that control protein trafficking and cell polarity. Among these PDZ proteins are LNXl and LNX2, E3 ubiquitin ligases which are known to control trafficking of transmembrane proteins. In this study we characterize the interaction between Frizzled-7 and LNX2. We demonstrate that LNX2 influences ubiquitylation of Frizzled-7 and has the ability to moderate signal transduction within the canonical Wnt pathway in breast cancer cells.

The Z-disc of striated muscle cells is a highly specialized three-dimensional structure which delineates the boundary of the individual sarcomeres. It accomplishes a unique role by anchoring actin filaments and acts as a molecular trigger for contraction. Beyond a well-defined structural role, in recent years it is emerging the hypothesis that Z-disc may be directly involved in the perception and transmission of muscular stress signals. To achieve these complex functions, many Z-disc proteins are involved in multiple protein interactions. The importance of these interactions is indicated by the fact that mutations in several Z-disc proteins can result in muscular dystrophies and/or cardiomyopathies in human and mice. The knowledge of Z-disc interactome and its regulation would improve by far the comprehension of the Z-disc biology and the onset of muscular disorders. The main goal of my project was to understand the complex network of protein-protein interactions occurring at the Z-disc of skeletal and cardiac muscle. In particular, my work was focused on two groups of Z-disc proteins: the FATZ and myotilin protein families on one hand, and some proteins belonging to the enigma family on the other hand. This work led to the identification of a specific interaction between the PDZ domains of enigma family members and the C-terminal five amino acids of the FATZ and myotilin families. The work of this thesis is part of a wider project involving the groups of Dr. G. Faulkner at ICGEB, Trieste, and Prof. O. Carpen at University of Turku, Finland. Together with our collaborators we noted that the C-terminal five amino acids of FATZ-1 (ETEEL), FATZ-2 (ESEDL), FATZ-3 (ESEEL), myotilin (ESEEL), palladin (ESEDL) and myopalladin (ESDEL) are highly similar. Searches in protein sequence database revealed that this E-[S/T]-[D/E]-[D/E]-L motif is restricted in Vertebrates to the FATZ and myotilin families of proteins, and it is evolutionary conserved from zebrafish to humans, indicating its importance for their biological function. The ELM program (a source for predicting functional sites in eukaryotic proteins) predicted that the terminal four amino acids of the FATZ family, myotilin, palladin and myopalladin constitute a binding motif for class III PDZ domain proteins (X-[D/E]-X-[V/I/L]). The first object of my work was to determine if the proteins with this new type of class III PDZ binding motif at their C-terminal could effectively bind PDZ domains. We knew from the literature that ZASP binds to all the three members of the FATZ family by means of its N-terminal PDZ domain, and that the C-terminal region of myotilin interacts with ZASP. In addition to ZASP, other two members of the enigma family of PDZ proteins, ALP and CLP-36, were included in this study. Both the full-length and the truncated (lacking the last five amino acids) version of the FATZ and myotilin families were produced as native proteins and tested for PDZ binding using the AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay) technique. Biotinylated phosphorylated and non-phosphorylated peptides corresponding to the C-terminal five amino acids of the FATZ family, myotilin, palladin and myopalladin were also used in AlphaScreen interaction experiments, as well as a control peptide having E instead of L as its last amino acid (ESEEE). The results presented in this thesis show that the final five amino acids of the FATZ and myotilin families of proteins are responsible for the binding to the PDZ domains of ZASP, ALP and CLP-36, and that the nature of the last amino acid of the motif is crucial for the interaction. We also show that phosphorylation of the ligand sequence modulates the ability of the peptides to bind to the PDZ domains of the enigma family. ?-actinin-2 was included in this study as its C-terminus (GESDL) is classified as a class I PDZ binding motif that is able to bind to ZASP and ALP PDZ domains. AlphaScreen experiments confirm the binding of both the full-length and the C-terminal phosphorylated and non-phosphorylated peptides of ?-actinin-2 to the PDZ domains of ZASP and ALP, and they also reveal an interaction with the PDZ domain of CLP-36. These interactions were verified using another in vitro binding technique, the TranSignal PDZ Domain Array. Based on the results of the PDZ arrays, RIL was found to be another member of the enigma family capable to bind to the E-[S/T]-[D/E]-[D/E]-L motif. Therefore, these final five amino acids can be considered a novel type of class III PDZ binding motif specific for the PDZ domains of enigma proteins. To better quantify the strength of the noted interactions, SPR (Surface Plasmon Resonance) experiments were performed in the laboratory of Dr. A. Baines at University of Kent, UK. The affinities of the interactions between the PDZ domain of ZASP and some of the phosphorylated and non-phosphorylated peptides of the FATZ and myotilin families result to be in the nM range. The SPR results also demonstrate a new interaction between the PDZ domain of ZASP and ANKRD2. This protein is a member of the MARP family and it is thought to be involved in muscle stress response pathways. ANKRD2 localizes both in the sarcomeric I-band and the nucleus, and it is able to bind to several transcription factors, including YB-1, PML and p53. This interaction strengthens the hypothesis that, besides a structural function, Z-disc could have a role in cell signalling. The fact that at the Z-disc many proteins can interact with the same partners, it would be helpful to define the pattern and level of expression of the individual proteins in different muscle tissues. Another aim of my work was to measure the abundance of mRNAs of some Z-disc proteins using the Real-Time PCR technique. Four different muscles from adult mice were considered: tibialis (a fast-twitch skeletal muscle), soleus (a slow-twitch skeletal muscle), gastrocnemius (a skeletal muscle with mixed fibers) and heart (cardiac muscle). The different distribution of the FATZ proteins, myotilin and the alternatively spliced variants of ZASP suggest that, at least in mouse, the interactions between these proteins could be compartmentalized in distinct fiber types.
Il disco-Z del muscolo striato è una struttura molecolare altamente specializzata a livello della quale si instaurano numerose interazioni proteina-proteina. Il disco-Z delinea il confine dei singoli sarcomeri, fornendo un punto di ancoraggio per i filamenti sottili di actina; il loro scorrimento sui filamenti spessi di miosina produce la forza meccanica responsabile della contrazione. Uno dei ruoli chiave del disco-Z, dunque, è quello di trasmettere la tensione generata dalla struttura seriale dei sarcomeri lungo le miofibrille e, di conseguenza, lungo tutto il muscolo. Al di là di un evidente significato strutturale, negli ultimi anni sta diventando sempre più consistente l’ipotesi di un suo coinvolgimento anche nella percezione e nella trasmissione di segnali. L’importanza delle interazioni tra le proteine del disco-Z è indicata dal fatto che mutazioni in molte di queste proteine possono risultare in distrofie muscolari e/o cardiomiopatie sia in uomo sia in topo. Una più ampia conoscenza delle interazioni che si articolano a livello del disco-Z e, in generale, degli eventi che le regolano, aiuterebbe a chiarire la biologia del disco-Z e l’insorgenza di eventuali patologie associate. Il mio progetto di Dottorato è stato incentrato su due gruppi di proteine sarcomeriche e sulle loro interazioni: le proteine delle famiglie FATZ e miotilina da un lato, e alcune proteine appartenenti alla famiglia enigma dall’altro. Questo lavoro ha portato all’identificazione di un’interazione specifica tra i domini PDZ delle proteine della famiglia enigma e gli ultimi cinque residui aminoacidici presenti nelle proteine delle famiglie FATZ e miotilina. Il lavoro di questa tesi fa parte di un progetto più ampio che coinvolge i gruppi coordinati dalla Dr.ssa G. Faulkner dell’ICGEB, Trieste, e il Prof. O. Carpen dell’Università di Turku, Finlandia. Grazie alla loro collaborazione, è stato possibile notare che i cinque residui C-terminali delle proteine FATZ-1 (ETEEL), FATZ-2 (ESEDL), FATZ-3 (ESEEL), miotilina (ESEEL), palladina (ESEDL) e miopalladina (ESDEL) sono molto simili. Una ricerca effettuata in database di sequenze proteiche ha rivelato che questo motivo, E-[S/T]-[D/E]-[D/E]-L, è quasi esclusivamente ristretto nei Vertebrati alle proteine delle famiglie FATZ e miotilina; inoltre, esso sembra essere conservato da zebrafish ad uomo, suggerendo la sua importanza per le proteine che lo contengono. Il programma ELM (che predice siti funzionali in proteine eucariotiche) ha predetto che gli ultimi quattro amino acidi delle proteine FATZ, miotilina, palladina e miopalladina costituiscono un motivo di legame per le proteine con domini PDZ di classe III (X-[D/E]-X-[V/I/L]). Il mio primo obiettivo è stato quello di verificare se le proteine caratterizzate da questo nuovo motivo C-terminale potessero effettivamente legare domini PDZ. E’ noto dalla letteratura che tutti e tre i componenti della famiglia FATZ legano il PDZ di ZASP, e che l’interazione tra ZASP e miotilina è mediata dalla regione C-terminale di quest’ultima. Oltre a ZASP, altri due membri della famiglia enigma, ALP e CLP-36, sono stati inclusi nello studio. Le proteine della famiglia FATZ e miotilina sono state prodotte sia in versione full-length sia priva degli ultimi cinque amino acidi per essere utilizzate in saggi di interazione AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay). Peptidi biotinilati, fosforilati e non, corrispondenti ai cinque amino acidi finali delle FATZ, miotilina, palladina e miopalladina sono stati inoltre impiegati nei saggi AlphaScreen, così come un peptide di controllo avente in ultima posizione un acido glutammico (E) invece che una leucina (L). I risultati riportati in questa tesi dimostrano che gli ultimi cinque amino acidi delle proteine delle famiglie FATZ e miotilina sono responsabili del legame ai domini PDZ di ZASP, ALP e CLP-36, e che la natura dell’ultimo residuo aminoacidico è cruciale per questa interazione. Inoltre, la fosforilazione del residuo di serina o treonina del ligando C-terminale può influenzare il legame dei peptidi nei confronti dei domini PDZ della famiglia enigma. La proteina ?-actinina-2 è stata introdotta nello studio, poiché la sua sequenza C-terminale (GESDL) è classificata come motivo di legame per i domini PDZ di classe I (X-[S/T]-X-[V/I/L]). Gli esperimenti AlphaScreen hanno confermato l’interazione di ?-actinina-2 (sia della forma full-length sia dei peptidi C-terminali, fosforilati e non) con i PDZ di ZASP e ALP, e hanno fatto emergere una nuova interazione con il PDZ di CLP-36. Molte di queste interazioni sono state verificate con un altro metodo di interazione proteina-proteina in vitro, il TranSignal PDZ Domain Array. Sulla base dei risultati di PDZ array è stato possibile identificare un altro membro della famiglia di proteine enigma, RIL, in grado di legare il motivo E-[S/T]-[D/E]-[D/E]-L. Possiamo considerare questi cinque amino acidi C-terminali come un nuovo motivo di legame per le proteine con domini PDZ di classe III, specifico per i domini PDZ delle proteine enigma. Per poter meglio quantificare la forza delle interazioni studiate, alcuni esperimenti di SPR (Surface Plasmon Resonance) sono stati eseguiti nel laboratorio del Dr. A. Baines all’Università di Kent, UK. Le affinità delle interazioni tra il dominio PDZ di ZASP e alcuni dei peptidi fosforilati e non-fosforilati delle famiglie di proteine FATZ e miotilina risultano essere nell’ordine del nM. Gli esperimenti di SPR hanno portato anche all’identificazione di un’interazione tra il PDZ di ZASP e ANKRD2. Si pensa che questa proteina, membro della famiglia MARP, sia coinvolta nelle vie di risposta a stress muscolari. ANKRD2 può trovarsi sia nella banda-I del sarcomero sia nel nucleo ed è in grado di legare diversi fattori di trascrizione, come YB-1, PML e p53. La scoperta di questa interazione rafforza l’ipotesi che il disco-Z, oltre ad un ruolo specificamente strutturale, potrebbe essere coinvolto in vie di segnalazione. Dal momento che a livello del disco-Z molte proteine hanno più di un partner proteico, sarebbe utile cercare di definire il livello e il profilo di espressione delle singole proteine in tessuti muscolari con diverse caratteristiche. Un altro obiettivo del mio lavoro è stato quindi quello di valutare l’abbondanza degli mRNA di alcune delle proteine del disco-Z da me studiate con la Real-Time PCR. Allo scopo sono stati presi in considerazione quattro tessuti muscolari di topo adulto: il tibiale (un muscolo scheletrico a contrazione rapida), il soleo (un muscolo scheletrico a contrazione lenta), il gastrocnemio (un muscolo scheletrico con fibre miste) e il muscolo cardiaco. La differente distribuzione delle FATZ, miotilina e ZASP (con le sue varianti di splicing) suggerisce che, almeno in topo, le interazioni tra queste proteine potrebbero essere compartimentalizzate in distinte fibre muscolari.

PSD-95/Dlg/ZO-1 (PDZ) domain - PDZ binding motif (PBM) interactions have been one of the most well studied protein-protein interaction systems through biochemical, biophysical and high-throughput screening (HTS) strategies. This has allowed us to understand the mechanism of individual PDZ-PBM interactions and the re-engineering of PBMs to bind tighter or to gain or lose certain specificity. However, there are several thousand native PDZ domains whose biological ligands remain unknown. Because of the low sequence identity among PDZ domain homologues, promiscuous binding profiles (defined as a PDZ domain that can accommodate a set of PBMs or a PBM that can be recognized by many PDZ domains), and context-dependent interaction mechanism, we have an inadequate understanding of the general molecular mechanisms that determine the PDZ-PBM specificity. Therefore, predicting PDZ specificity has been elusive. In addition, no de novo PBM ligand or artificial non-native PDZ domain have been successfully designed. This reflects the general challenges in understanding the general principles of PDZ-ligand interactions, namely that they are context-dependent, exhibit weak binding affinity, narrow binding energy range, and larger interaction surface than other protein-ligand interactions. Together, PDZ domains make good model systems to investigate the fundamental principles of protein-protein interactions with a wide spectrum of biomedical implications. My studies suggest that understanding PBM specificity with the set of structural positions forming the binding pocket can connect sequence, structure and function of a PDZ domain in a general context. They also suggest that this way of understanding the specificity will shed light on prediction and engineering of specificity rationally. Structural analysis on most of the available PDZ domain structures was established to support the principle (Chapter I). The principle was tested against two different types of PBM; C-terminal PBM (Chapter II) and internal PBM (Chapter III), and shown to support better understanding and design of PDZ domain specificity. We further applied the principle to design de novo PDZ domains, and the preliminary data hints that it is optimistic to engineer PDZ domain specificity (Appendix A and B).

The work presented in this thesis has contributed with knowledge to several aspects of protein-protein interaction involving PDZ domains. A substantial amount of our proteome contains regions that are intrinsically disordered but fold upon ligand interaction. The mechanism by which disordered regions bind to their ligands is one important piece of the puzzle to understand why disorder is beneficial. A region in the PDZ domain of nNOS undergoes such a disorder-to-order transition to form a b-sheet in the binding pocket of its partner. By studying the kinetics of interaction, in combination with mutations that modulate the stability of the aforementioned region, we demonstrate that the binding mechanism consists of multiple steps in which the native binding interactions of the b-sheet are formed cooperatively after the rate-limiting transition state. These mechanistic aspects may be general for the binding reactions of intrinsically disordered protein regions, at least upon formation of β-sheets.               The second part of this thesis deals with the engineering of proteins for increasing affinity in protein-protein interaction. Infection by high-risk human papillomavirus (hrHPV) can lead to cancer, and the viral E6 protein is an attractive drug target. E6 from hrHPV natively interacts with the well-characterized PDZ2 domain in SAP97, which we used as a scaffold to develop a high affinity bivalent binder of hrHPV E6. We initially increased PDZ2's affinity for E6 6-fold, but at the cost of decreased specificity. Attaching a helix that binds E6 at a distant site, increasing the affinity another14-fold, completed the design.             The final work of this thesis investigates if binding studies conducted with isolated PDZ domains is representative of the full-length proteins they belong to. It has been suggested that ligand binding in PDZ domains can be influenced by factors such as adjacent domains and interactions outside of the binding pocket. We studied these aspects for the three PDZ domains of PSD-95 and found that they on the whole function in an independent manner with short peptides as ligands, but that interactions outside of the PDZ binding-pocket may be present. The representative length of the PDZ interaction partner should therefore be considered.

Les interactions protéine-protéine sont souvent contrôlées par de petits domaines protéiques qui régulent les chemins de signalisation au sein des cellules eucaryotes. Les domaines PDZ sont parmi les domaines les plus répandus et les plus étudiés. Ils reconnaissent spécifiquement les 4 à 10 acides aminés C-terminaux de leurs partenaires. Tiam1 est un facteur d'échange de GTP de la protéine Rac1 qui contrôle la migration et la prolifération cellulaire et dont le domaine PDZ lie les protéines Syndecan-1 (Sdc1), Caspr4 et Neurexine. Des petits peptides ou des molécules peptidomimétiques peuvent potentiellement inhiber ou moduler son activité et être utilisés à des fins thérapeutiques. Nous avons appliqué des approches de dessin computationnel de protéine (CPD) et de calcul d'énergie libre par simulations dynamique moléculaire (DM) pour comprendre et modifier sa spécificité. Le CPD utilise un modèle structural et une fonction d'énergie pour explorer l'espace des séquences et des structures et identifier des variants protéiques ou peptidiques stables et fonctionnels. Nous avons utilisé le programme de CPD Proteus, développé au laboratoire, pour redessiner entièrement le domaine PDZ de Tiam1. Les séquences générées sont similaires à celles des domaines PDZ naturels, avec des scores de similarité et de reconnaissance de pli comparables au programme Rosetta, un outil de CPD très utilisé. Des séquences contenant environ 60 positions mutées sur 90, ont été testées par simulations de DM et des mesures biophysiques. Quatre des cinq séquences testées expérimentalement (par nos collaborateurs) montrent un dépliement réversible autour de 50°C. Proteus a également déterminer correctement la spécificité de la liaison de quelques variants protéiques et peptidiques. Pour étudier plus finement la spécificité, nous avons paramétré un modèle d'énergie libre semi-empirique de Poisson-Boltzmann ayant la forme d'une énergie linéaire d'interaction, ou PB/LIE, appliqué à des conformations issues de simulations de DM en solvant explicite de complexes PDZ:peptide. Avec trois paramètres ajustables, le modèle reproduit correctement les affinités expérimentales de 41 variants, avec une erreur moyenne absolue de 0,4~kcal/mol, et donne des prédictions pour 10 nouveaux variants. Le modèle PB/LIE a ensuite comparé à la méthode non-empirique de calcul d'énergie libre par simulations alchimiques, qui n'a pas de paramètre ajustable et qui prédit correctement l'affinité de 12 complexes Tiam1:peptide. Ces outils et les résultats obtenus devraient nous permettre d'identifier des peptides inhibiteurs et auront d'importantes retombées pour l'ingénierie des interactions PDZ:peptide
Small protein domains often direct protein-protein interactions and regulate eukaryotic signalling pathways. PDZ domains are among the most widespread and best-studied. They specifically recognize the 4-10 C-terminal amino acids of target proteins. Tiam1 is a Rac GTP exchange factor that helps control cellmigration and proliferation and whose PDZ domain binds the proteins syndecan-1 (Sdc1), Caspr4, and Neurexin. Short peptides and peptidomimetics can potentially inhibit or modulate its action and act as bioreagents or therapeutics. We used computational protein design (CPD) and molecular dynamics (MD) free energy simulations to understand and engineer its peptide specificity. CPD uses a structural model and an energy function to explore the space of sequences and structures and identify stable and functional protein or peptide variants. We used our in-house Proteus CPD package to completely redesign the Tiam1 PDZ domain. The designed sequences were similar to natural PDZ domains, with similarity and fold recognition scores comarable to the widely-used Rosetta CPD package. Selected sequences, containing around 60 mutated positions out of 90, were tested by microsecond MD simulations and biophysical experiments. Four of five sequences tested experimentally (by our collaborators) displayed reversible unfolding around 50°C. Proteus also accurately scored the binding specificity of several protein and peptide variants. As a more refined model for specificity, we parameterized a semi-empirical free energy model of the Poisson-Boltzmann Linear Interaction Energy or PB/LIE form, which scores conformations extracted from explicit solvent MD simulations of PDZ:peptide complexes. With three adjustable parameters, the model accurately reproduced the experimental binding affinities of 41 variants, with a mean unsigned error of just 0.4 kcal/mol, andgave predictions for 10 new variants. The PB/LIE model was tested further by comparing to non-empirical, alchemical, MD free energy simulations, which have no adjustable parameters and were found to give chemical accuracy for 12 Tiam1:peptide complexes. The tools and insights obtained should help discover new tight binding peptides or peptidomimetics and have broad implications for engineering PDZ:peptide interactions

Proteins present at the synapse need to be multitasking in order to perform all vital functions in this limited space. In this thesis I have analyzed the function and evolution of such proteins, focusing on the PDZ domain and the paralemmin family. The PDZ domains bind to a wide variety of interaction partners. The affinity for each partner is regulated by residues at the binding site, but also through intradomain allostery. How this intradomain allostery is transferred to the binding site is not established. I here show that side chain interactions can explain all transfer of intradomain allostery in three analyzed PDZ domains. A circularly permuted PDZ domain has an identical set of amino acids as the original protein and a very similar structure with only a few perturbed side chains. By using the circular permutant I show that a slight alteration in the position of a side chain leads to a corresponding change in allosteric signal. I further study the folding of several PDZ domains and show that they all fold via a conserved folding mechanism, supporting the notion that the final structure has a part in deciding folding mechanism. The folding mechanism of the circularly permuted PDZ domain is conserved compared to the original protein illustrating how circular permutations can be tolerated through evolution. The multifunctionality of paralemmins probably lies in their highly flexible structures. I have studied the evolution of the paralemmins and found that the four mammalian paralemmins arose in the two whole-genome duplications that occurred early in the vertebrate evolution. The fact that all four paralemmins have survived evolution since the gene duplications suggests that they have important functions, possibly in the development of the nervous system. Synaptic proteins are crucial for many biological processes, and their misfolding implicated in many diseases. The results presented here shed light on the mechanisms of action of the synaptic proteins and will help us to understand how they generate disease.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes
Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions

Le « Computational protein design » ou CPD est la recherche des séquences d’acides aminés compatibles avec une structure protéique ciblée. L’objectif est de concevoir une fonction nouvelle et/ou d’ajouter un nouveau comportement. Le CPD est en développement dans de notre laboratoire depuis plusieurs années, avec le logiciel Proteus qui a plusieurs succès à son actif.Notre approche utilise un modèle énergétique basé sur la physique et s’appuie sur la différence d’énergie entre l’état plié et l’état déplié de la protéine. Au cours de cette thèse, nous avons enrichi Proteus sur plusieurs points, avec notamment l’ajout d’une méthode d’exploration Monte Carlo avec échange de répliques ou REMC. Nous avons comparé trois méthodes stochastiques pour l’exploration de l’espace de la séquence : le REMC, le Monte Carlo simple et une heuristique conçue pour le CPD, le «Multistart Steepest Descent » ou MSD. Ces comparaisons portent sur neuf protéines de trois familles de structures : SH2, SH3 et PDZ. En utilisant les techniques d’exploration ci-dessus, nous avons été en mesure d’identifier la conformation du minimum global d’énergie ou GMEC pour presque tous les tests dans lesquels jusqu’à 10 positions de la chaîne polypeptidique étaient libres de muter (les autres conservant leurs types natifs). Pour les tests avec 20 positions libres de muter, le GMEC a été identifié dans 2/3 des cas. Globalement, le REMC et le MSD donnent de très bonnes séquences en termes d’énergie, souvent identiques ou très proches du GMEC. Le MSD a obtenu les meilleurs résultats sur les tests à 30 positions mutables. Le REMC avec huit répliques et des paramètres optimisés a donné le plus souvent le meilleur résultat lorsque toutes les positions peuvent muter. De plus, comparé à une énumération exacte des séquences de faible énergie, le REMC fournit un échantillon de séquences de grande diversité.Dans la seconde partie de ce travail, nous avons testé notre modèle pour la conception de domaines PDZ. Pour l’état plié,nous avons utilisé deux variantes d’un modèle de solvant GB. La première utilise une frontière diélectrique protéine/solvant effective moyenne ; la seconde, plus rigoureuse, utilise une frontière exacte qui fluctue le long de la trajectoire MC. Pour caractériser l’état déplié, nous utilisons un ensemble de potentiels chimiques d’acide aminé ou énergies de références. Ces énergies de références sont déterminées par maximisation d’une fonction de vraisemblance afin de reproduire les fréquences d’acides aminés des domaines PDZ naturels. Les séquences conçues par Proteus ont été comparées aux séquences naturelles. Nos séquences sont globalement similaires aux séquences Pfam, au sens des scoresBLOSUM40, avec des scores particulièrement élevés pour les résidus au cœur de la protéine. La variante de GB la plus rigoureuse donne toujours des séquences similaires à des homologues naturels modérément éloignés et l’outil de reconnaissance de plis Super family appliqué à ces séquences donne une reconnaissance parfaite. Nos séquences ont également été comparées à celles du logiciel Rosetta. La qualité, selon les mêmes critères que précédemment, est très comparable, mais les séquences Rosetta présentent moins de mutations que les séquences Proteus
Computational Protein Design, or CPD is the search for the amino acid sequences compatible with a targeted protein structure. The goal is to design a new function and/or add a new behavior. CPD has been developed in our laboratory for several years, with the software Proteus which has several successes to its credit. Our approach uses a physics-based energy model, and relies on the energy difference between the folded and unfolded states of the protein. During this thesis, we enriched Proteus on several points, including the addition of a Monte Carlo exploration method with Replica Exchange or REMC. We compared extensively three stochastic methods for the exploration of sequence space: REMC, plain Monte Carlo and a heuristic designed for CPD: Multistart Steepest Descent or MSD.These comparisons concerned nine proteins from three structural families: SH2, SH3 and PDZ. Using the exploration techniques above, we were able to identify the Global Minimum EnergyConformation, or GMEC for nearly all the test cases where up to10 positions of the polypeptide chain were free to mutate (the others retaining their native types). For the tests where 20positions were free to mutate, the GMEC was identified in 2/3 of the cases. Overall, REMC and MSD give very good sequences in terms of energy, often identical or very close to the GMEC. MSDperformed best in the tests with 30 mutating positions. REMCwith eight replicas and optimized parameters often gave the best result when all positions could mutate. Moreover, compared to an exact enumeration of the low energy sequences, REMC provided a sample of sequences with a high sequence diversity.In the second part of this work, we tested our CPD model forPDZ domain design. For the folded state, we used two variants ofa GB solvent model. The first used a mean, effective protein/solvent dielectric boundary; the second one, more rigorous, used an exact boundary that flucutated over the MCtrajectory. To characterize the unfolded state, we used a set of amino acid chemical potentials or reference energies. These reference energies were determined by maximizing a likelihoodfunction so as to reproduce the amino acid frequencies in naturalPDZ domains. The sequences designed by Proteus were compared to the natural sequences. Our sequences are globally similar to the Pfam sequences, in the sense of the BLOSUM40scores, with especially high scores for the residues in the core ofthe protein. The more rigorous GB variant always gives sequences similar to moderately distant natural homologues and perfect recognition by the the Super family fold recognition tool.Our sequences were also compared to those produced by the Rosetta software. The quality, according to the same criteria as before, was very similar, but the Rosetta sequences exhibit fewer mutations than the Proteus sequences

Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles. In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions. I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding. Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction. Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

Il existe de nombreux mécanismes impliqués dans le développement des tissus qui nécessitent que des cellules ou des groupes de cellules s’orientent et se polarisent. Les protéines de la voie de la polarité planaire (PP) s’associent pour former des complexes à la membrane et créer des asymétries proximo-distale. Vangl2 et Scrib1 ont été identifiés comme les deux premiers gènes impliqués dans la PP chez les mammifères. Lors de ma thèse, je me suis intéressé à ces deux protéines et à certains des complexes dans lesquelles elles sont impliquées. Dans un premier temps, nous avons montré l’implication directe de Scribble1 dans le trafic après endocytose des récepteurs NMDA. Scrib1 interagit avec les récepteurs NMDA grâce à ses domaines PDZ. Scribble1 peut interagir avec le complexe AP2 qui intervient dans l’endocytose des récepteurs. Cette étude a permis de définir un nouveau mécanisme dans lequel Scrib1 régule la quantité de récepteurs NMDA à la membrane et donc participe à la plasticité synaptique. Vangl2 est l’une des protéines transmembranaires les plus en amont de la voie de la PP. Nous avons identifié un nouveau partenaire nommé "Axin Interaction partner and Dorsalization Antagonist" (AIDA). Nous avons montré, par double hybride en levure et pull down, l’interaction de Vangl2 avec les deux isoformes de AIDA et leur colocalisation en COS7 et neurones. Ensemble, ces données présentent AIDA comme un très bon candidat pour le maintien de Vangl2 aux jonctions adhérentes et/ou pour son adressage à la membrane. Ces études nous ont permis d’améliorer notre compréhension des mécanismes impliquant les protéines de la polarité planaire
There are many mechanisms involved in the development of tissues that require cells or groups of cells orient and polarize. The proteins of the planar cell polarity (PCP) combine to form complexes with the membrane and create proximal-distal asymmetries. Vangl2 and Scrib1 have been identified as the first two genes involved in the PCP in mammals. In this study, I am interested in these two proteins and some of the complex in which they are involved. At first, using techniques of biochemistry, cell biology and biophysics, we showed the direct involvement of Scribble1 in traffic after endocytosis of NMDA receptors. Scrib1 interacts with NMDA receptors through its PDZ domains. Due to this binding motif between PDZ1 and PDZ2 of Scrib1, it can interact with the AP2 complex which is involved in receptor endocytosis. This study has identified a new mechanism in which Scrib1 regulates the amount of NMDA receptors on the membrane and is therefore involved in synaptic plasticity. Vangl2 is a transmembrane protein of the most upstream of the PCP pathway. We have identified a new partner named "Axin Interaction partner and Dorsalization Antagonist" (AIDA). We have shown, by yeast two-hybrid and pull down the interaction of Vangl2 with two isoforms of AIDA and collocation in COS7 and neurons. Together, these data show AIDA as a very good candidate for maintaining Vangl2 to adherens junctions and/or its membrane targeting. These studies have allowed us to improve our understanding of the mechanisms involving the planar polarity proteins

Abstract The correct three dimensional structures of proteins are essential for their ability to function properly. Proteins start to fold as soon as they are synthesized in the ribosomes from activated amino acids. Many secreted, cell-surface, secretory pathway and endoplasmic reticulum (ER) lumenal proteins have in their amino acid sequence cysteine residues which form intra- and intermolecular disulfide bridges that stabilize the overall fold of the proteins and protein complexes. The formation of correct disulfide bonds is a complex process which takes place within the ER. Protein disulfide isomerase (PDI) is the key enzyme in the formation and rearrangement of correct disulfide bonds in the ER. It is an archetypal and the best studied member of the PDI family, i.e. a group of ER proteins that resemble thioredoxin (TRX), a protein reductase, in their structure. PDI has a four domain a-b-b'-a' structure the a and a' domains having the catalytic activity and amino acid sequence similarity to TRX. In addition to its function as a thiol-disulfide oxidoreductase, PDI acts as the β subunit in two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein (MTP). The closest homologue of PDI is the multifunctional enzyme and chaperone ERp57 that functions in concert with two lectins, calnexin (CNX) and calreticulin (CRT) specifically in the folding of proteins that have sugar moieties linked to them. ERp57 is 56% similar to PDI in its amino acid sequence and has also the four-domain architecture. Despite the high similarity in their structures ERp57 cannot substitute for PDI as the β subunit of C-P4H. The minimum requirement for the C-P4H tetramer assembly is fulfilled by domains b' and a' of PDI, while domains a and b enhance this function and can be substituted in part by those of ERp57. Until very recently the structural information of any of the PDI family members, which contains the TRX active site was limited to solution structures of human PDI domains a and b. In this research the domain boundaries of the full length ERp57 were defined and the individual domains characterized. Furthermore the solution structures of the catalytically active domains a and a' of ERp57 were studied by nuclear magnetic resonance (NMR).

Les protéines à domaine PDZ, en très grand nombre dans le génome humain, sont impliquées dans des interactions protéine-protéine. Elles participent ainsi à véhiculer des signaux à l'origine de différentes pathologies (cancer, douleur....). L'interruption de l'interaction entre la protéine à domaine PDZ, PSD-95, et le récepteur de la sérotonine, 5-HT2A, entraîne une réduction de l'hyperalgie chez le rat neuropathique. Le développement de molécules capables d'inhiber cette interaction pourrait donc conduire à une nouvelle classe d'antalgiques.Nous avons réalisé, au cours de ces travaux, la synthèse de trois générations de ligands, comportant un noyau indolique, capables d'interagir avec le site S0, site très conservé des protéines à domaines PDZ. Dans un premier temps, nous avons préparé 15 biligands possédant un noyau indolique polysubstitué lié, via un espaceur de longueur variable (2 à 6 atomes de carbone), à différents acides aminés, dans le but d'interagir avec le site S1, montrant beaucoup de diversité en fonction du domaine. Nous avons ensuite, après une étude de relation structure/activité, développé deux autres générations d'indoles polysubstitués présentant notamment des substituants hydrophobes en position 5.Nous avons montré, par RMN HSQC 1H/15N et chromatographie d'affinité, que deux de ces composés sont des inhibiteurs de l'interaction PSD-95/5-HT2A et présentent de fortes interactions avec le site S0 de PSD-95. Ces molécules présentent également des propriétés antalgiques particulièrement intéressantes in vivo. Nous avons également déterminé, par RMN NOESY, la structure du complexe protéine/ligand pour ces deux composés. L'orientation d'une de ces molécules dans le site de la protéine nous permet d'envisager le développement d'une nouvelle génération d'indoles polysubstitués, pouvant interagir avec le site S1 de la protéine et permettant ainsi d'obtenir des inhibiteurs sélectifs de l'interaction PSD-95/5-HT2A.

Diacylglycerol kinase-zeta (DGK-zeta) attenuates diacylglycerol (DAG) signaling by converting it into phosphatidic acid (PA). DGK-zeta binds via its C-terminus, which contains a PSD-95/ Discs-Large/ZO-1 (PDZ)-binding motif, to a family of PDZ domain-containing scaffold proteins called syntrophins. Previous studies showed that some PDZ-mediated interactions are regulated by phosphorylation of the C-terminal PDZ-binding motif, however, to my knowledge, there are no published reports which demonstrate that a phosphorylation outside of this motif regulates PDZ interactions. Here, I provide evidence that protein kinase C-mediated phosphorylation of the MARCKS domain of DGK-zeta increases its association with syntrophins by a PDZ-dependent mechanism. Compared to wild-type (wt) DGK-zeta, a mutant mimicking phosphorylation of the MARCKS domain (DGK-zeta M1) bound more to recombinant syntrophin PDZ domains in in vitro binding assays. Moreover, more endogenous syntrophin co-immunoprecipitated from lysates of COS cells infected with HA-tagged DGK-zetaM1 than with wt HA-DGK-zeta. Protein kinase C (PKC) activation by phorbol myristate acetate enhanced the interaction of wt DGK-zeta and syntrophin PDZ domains, an effect that was blocked by a specific PKC inhibitor. Consistent with the idea that the MARCKS domain regulates binding, deletion of this domain decreased binding to syntrophin PDZ domains. Surprisingly, phosphorylation-mimicking mutants of extracellular signal-regulated kinase phosphorylation sites closer to the C-terminus had no detectable effect on syntrophin binding. Collectively, my findings suggest PKC-mediated phosphorylation of the MARCKS domain regulates the PDZ-dependent interaction between DGK-zeta and syntrophins.

Cancer has been a pervasive and deadly problem for many years. No treatments have been developed that effectively destroy cancer cells while also keeping healthy cells safe. In this work, the knob-socket construct is used to analyze two systems involved in cancer pathways, the PDZ domain and the Bcl-BH3 complex. Application of the knob-socket model in mapping the packing surface topology (PST) allows a direct analysis of the residue groups important for peptide specificity and affinity in both of these systems. PDZ domains are regulatory proteins that bind the C-terminus of peptides involved in the signaling pathway of cancer progression. The domain includes five -strands, two -helices, and six coils/turns. In this study, the PST of all eight solved crystal structures of T-cell lymphoma invasion and metastasis 1 (Tiam1) PDZ domains are mapped to reveal details of ligand-domain binding pockets and packing interactions. Four main interactions were identified in the comparison of the PST maps and a consensus sequence was calculated using knob-socket interaction data. In the case of the Bcl-BH3 complex, binding of these two proteins prevents an unhealthy cell from undergoing apoptosis. In the knob-socket mapped protein-ligand interactions, the helical ligand consists of 8 to 10 residues that specifically interact with four helices on the binding protein: the N-terminus of Helix2, the main bodies of Helix3 and Helix4 and the C-terminus of Helix5. Among all of the interactions that were analyzed, there were three amino acids from the ligand, glycine, leucine, and isoleucine, that always packed into the hydrophobic groove that is key for ligand recognition. By using knob-socket analysis to map quaternary packing structure, it was possible to identify the quaternary-level protein interactions that define ligand specificity and binding strength. From this analysis, possible protein mimetics can be developed that could be used as cancer treatments.

At the time of writing this thesis, the complete genomes of more than 180 organisms have been sequenced and more than 80000 biological macromolecular structures are available in the Protein Data Bank (PDB). While the number of sequenced genomes and solved three-dimensional structures are rapidly increasing, the functional annotation of protein sequences and structures is a much slower process, mostly because the experimental de-termination of protein function is expensive and time-consuming. A major class of in silico methods used for protein function prediction aim to transfer annotations between proteins based on sequence or structural similarities. These approaches rely on the assumption that homologous proteins of similar primary sequences and three-dimensional structures also have similar functions. While in most cases this assumption appears to be valid, an increasing number of examples show that proteins of highly similar sequences and/or structures can have different biochemical functions. Thus the relationship between the divergence of protein sequence, structure and function is more complex than previously anticipated. On the other hand, there is mounting evidence suggesting that minor changes of the sequences and structures of proteins can cause large differences in their conformational dynamics. As the intrinsic fluctuations of many proteins are key to their biochemical functions, the fact that very similar (almost identical) sequences or structures can have entirely different dynamics might be important for understanding the link between sequence, structure and function. In other words, the dynamic similarity of proteins could often serve as a better indicator of functional similarity than the similarity of their sequences or structures alone. Currently, little is known about how proteins are distributed in the 'dynamics space' and how protein motions depend on structure and sequence. These problems are relevant in the field of protein design, studying protein evolution and to better understand the functional differences of proteins. To address these questions, one needs a precise definition of dynamic similarity, which is not trivial given the complexity of protein motions. This thesis is intended to explore the possibilities of describing the similarity of proteins in the 'dynamics space'. To this end, novel methods of characterizing and comparing protein motions based on molecular dynamics simulation data were introduced. The generally applicable approach was tested on the family of PDZ domains; these small protein-protein interaction domains play key roles in many signalling pathways. The methodology was successfully used to characterize the dynamic dissimilarities of PDZ domains and helped to explain differences of their functional properties (e.g. binding promiscuity) also relevant for drug design studies. The software tools developed to implement the analysis are also introduced in the thesis. Finally, a network analysis study is presented to reveal dynamics-mediated intramolecular signalling pathways in an allosteric PDZ domain.

Contrairement à celui des animaux, le développement des plantes est pour une grande part post-embryonnaire et beaucoup plus influencé par l'environnement. Ceci suggère que les plantes ont évolué en élaborant différents mécanismes pour contrôler leur développement et leur croissance. La division cellulaire joue un rôle essentiel dans le développement et la croissance de la plante. Durant cette division, la transition G1-S est une étape décisive vers la prolifération cellulaire ou la différenciation. Par des approches génétiques, physiologiques et moléculaires, nous avons montré que Nicta;CYCA3;2 a des fonctions importantes, d’une maniére analogue à la cycline E (un régulateur essentiel à la transition G1/S) chez les animaux, dans le contrôle de la division et différentiation cellulaire. Une fois la phase S initiée, l'ADN soit répliqué et la chromatine soit assemblé. La mise en place et la conservation d'états spécifiques de la chromatine fait appel à un mécanisme épigénétique pour assurer la transmission aux cellules filles d’une identité similaire de la cellule mère. Nos travaux sur NtSET1, une protéine à domaine SET chez le tabac, ont montré que cette protéine est capable de méthyler H3K9. L'expression ectopique de NtSET1 augmente la diméthylation de H3K9 et induit des défauts de ségrégation chromatinienne dans les cellules du tabac BY2. En plus, j'ai mis en évidence une interaction protéique entre NtSET1 et la protéine LHP1, l'homologue unique d'Arabidopsis de HP1 chez les animaux. Par l'immunolocalisation et l'identification des cibles ADN, j'ai montré que NtSET1-YFP et LHP1-YFP lient des régions hétérochromatiniennes, suggérant une implication dans la formation d'hétérochromatine. L’étude du gène SDG8 chez l’Arabidopsis a montré que SDG8 code une enzyme importante dans le contrôle de la méthylation en H3K36 chez l’Arabidopsis, qui régule positivement la transcription de FLC pour empêcher une floraison précoce. En conclusion, l’ensemble de mes travaux de thèse m’avaient permis d’acquérir une bonne connaissance en développement de plantes. Les connaissances et techniques acquises en biologie moléculaire et cellulaire me permettraient non seulement d’envisager une poursuite dans la recherche végétale mais également de s’adapter avec facilité à une recherche sur un autre organisme
The development of plants, compared with that of animals, is largely post-embryonic and influenced much more by the environment, suggesting that the plants have evolved and distinct mechanisms to control plant development and growth. Cell division plays a vital role in plant development and growth. During the cell division, the G1-S transition is a crucial crossroad at the interface between cell proliferation and differentiation. By genetic, physiological, and molecular approaches, we demonstrate that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E (an important regulator involved in G1-S transition) in animals, in the control of plant cell division and differentiation. Once S phase is initiated, DNA is replicated and chromatin is assembled. The establishment and maintenance of specific chromatin states provides an epigenetic mechanism for inheriting expression states throughout plant development. Our studies show that the tobacco SET domain protein NtSET1 methylates H3K9, which marks primarily heterochromatin. Ectopic expression of NtSET1 increases the amount of H3K9 dimethylation and induces chromosome segregation defects in tobacco BY2 cells. Furthermore, NtSET1 is shown to bind LHP1, the only Arabidopsis homologue of animal heterochromatin protein 1. By immunolocalization and in vivo target analysis, it shows that both NtSET1-YFP and LHP1-YFP bind heterochromatic regions, suggesting a mechanism of function in heterochromatin formation. Study of Arabidopsis SET domain group gene SDG8 reveals that SDG8 encodes a major enzyme controlling methylation of H3K36 in Arabidopsis, which positively regulates FLC transcription to prevent early flowering. In conclusion, my thesis work allows me to acquire a better understanding of plant development. The knowledge and techniques acquired in molecular and cellular biology will not only enable me to continue the research in plants but also adapt with the research on other organisms