La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulation. / Oxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect.

La sélénocystéine est incorporée co-traductionnellement dans les sélénoprotéines en réponse à un codon UGA habituellement l'un des 3 codons stop. La protéine SBP2 joue un rôle majeur dans ce mécanisme de recodage en se liant à une structure en tige-boucle (SECIS) située dans la région 3'UTR de l'ARNm des sélénoprotéines. Nous avons isolé et caractérisé fonctionnellement SBP2 de Drosophila melanogaster. Par comparison avec SBP2 humaine, nous avons identifié un domaine de liaison à l'ARN additionnel essentiel à la liaison au SECIS et à la sous-unité ribosomique 60S et permettant une sélectivité structurale du SECIS. Des prédictions structurales et des analyses biophysiques ont établi que SBP2 était une protéine globalement désordonnée ou “Intrinsically Disordered Protein” qui ne se replie qu'en présence de partenaires. Enfin, nous avons établi que l'assemblage des mRNP de sélénoprotéines faisait appel à des facteurs communs et présentait de multiples similarités avec celui des sn/snoRNP.

Proteins are the fundamental macromolecules within a cell that carry out most of the biological functions. The computational study of protein structure and its functions, using machine learning and data analytics, is elemental in advancing the life-science research due to the fast-growing biological data and the extensive complexities involved in their analyses towards discovering meaningful insights. Mapping of protein’s primary sequence is not only limited to its structure, we extend that to its disordered component known as Intrinsically Disordered Proteins or Regions in proteins (IDPs/IDRs), and hence the involved dynamics, which help us explain complex interaction within a cell that is otherwise obscured. The objective of this dissertation is to develop machine learning based effective tools to predict disordered protein, its properties and dynamics, and interaction paradigm by systematically mining and analyzing large-scale biological data. In this dissertation, we propose a robust framework to predict disordered proteins given only sequence information, using an optimized SVM with RBF kernel. Through appropriate reasoning, we highlight the structure-like behavior of IDPs in disease-associated complexes. Further, we develop a fast and effective predictor of Accessible Surface Area (ASA) of protein residues, a useful structural property that defines protein’s exposure to partners, using regularized regression with 3rd-degree polynomial kernel function and genetic algorithm. As a key outcome of this research, we then introduce a novel method to extract position specific energy (PSEE) of protein residues by modeling the pairwise thermodynamic interactions and hydrophobic effect. PSEE is found to be an effective feature in identifying the enthalpy-gain of the folded state of a protein and otherwise the neutral state of the unstructured proteins. Moreover, we study the peptide-protein transient interactions that involve the induced folding of short peptides through disorder-to-order conformational changes to bind to an appropriate partner. A suite of predictors is developed to identify the residue-patterns of Peptide-Recognition Domains from protein sequence that can recognize and bind to the peptide-motifs and phospho-peptides with post-translational-modifications (PTMs) of amino acid, responsible for critical human diseases, using the stacked generalization ensemble technique. The involved biologically relevant case-studies demonstrate possibilities of discovering new knowledge using the developed tools.

ErbB2/HER2 est un récepteur tyrosine kinase de la famille d'EGFR (ErbB1) surexprimé dans plus de 20% des cancers du sein et associé à une forme particulièrement agressive de la maladie. Les récepteurs ErbBs sont actifs seulement sous forme de dimères, permettant la phosphorylation de leur queue C-terminale par leur domaine tyrosine kinase. La phosphorylation entraine l'interaction avec des protéines adaptatrices et l'activation de voies de signalisation, Ras/MAPK et PI3K/Akt principalement. Ces voies contrôlent la prolifération, la motilité cellulaire et la résistance à l'apoptose. Contrairement à ErbB1/3/4, ErbB2 dimérise en l'absence de ligand. Comprendre les autres mécanismes de régulation de la phosphorylation de ses tyrosines et de ses interactions est donc particulièrement intéressant.ErbB2 a fait l'objet de nombreuses études structurales et fonctionnelles. Elles ont permis la mise au point de traitements ciblés efficaces mais sujets à l'apparition de résistance, dont l'anticorps Trastuzumab, ciblant sa partie extracellulaire. La queue C-terminale d'ErbB2 (CtErbB2) a été très souvent ignorée dans ces études. Cette partie étant intrinsèquement désordonnée, il a fallu attendre ces dernières années pour que les concepts et les outils permettant de l'étudier émergent.Dans cette thèse, j'ai d'abord effectué la caractérisation structurale et dynamique de CtErbB2. J'ai montré que bien qu'étant dépourvue de toute structure stable, cette région riche en prolines possède plusieurs structures secondaires transitoires et un contact longue-distance participant très probablement à la régulation de ses interactions intra- et inter-moléculaires. Dans une deuxième partie je me suis intéressée à la caractérisation de la protéine adaptatrice Grb2, partenaire essentiel de ErbB2 pour l'activation de la voie des MAP kinases. L'organisation en solution des domaines de cette protéine modulaire dans sa forme libre était jusque là inconnue. J'ai ensuite étudié l'interaction entre Grb2 et CtErbB2, et montré que CtErbB2 interagit non seulement avec le domaine SH2 de Grb2 (par l'intermédiaire d'une phosphotyrosine), mais aussi avec son domaine SH3 N-terminal (grâce à un motif polyproline). Enfin, j'ai mis en place plusieurs stratégies de phosphorylation des tyrosines de CtErbB2, dans le but d'étudier plus largement l'effet des phosphorylations sur l'ensemble de cette région. / ErbB2/HER2 is a receptor tyrosine kinase of the EGFR (ErbB1) family overexpressed in 20% of breast cancers and associated to a particularly aggressive form of the disease. ErbB receptors are only active upon dimerization that enables phosphorylation of their C-terminal tail by their tyrosine kinase domain. Phosphorylation then triggers interaction with adaptor proteins and activation of signaling pathways, mainly Ras/MAPK and Akt/PI3K. Those pathways control cell proliferation, motility and resistance to apoptosis. Contrary to ErbB1/3/4, ErbB2 can dimerize without any ligand. Understanding other mechanisms of regulation of its tyrosine phosphorylation and of its interactions is thus particularly interesting.ErbB2 structure and function have been extensively studied. This has led to the development of several FDA-approved targeted drugs, that are effective but to which resistance occurs, amongst which the Trastuzumab antibody that targets ErbB2 extracellular domain. The C-terminal tail of ErbB2 (CtErbB2) has been widely ignored in these studies. Since it is intrinsically disordered, the concepts and tools to study it have only emerged in the last few years.In the present work, I have performed the structural and dynamic study of CtErbB2. I showed that despite its lack of any stable structure, this proline-rich region exhibits several transient secondary structures and a long-range contact that might participate in the regulation of its intra- and inter-molecular interactions. Then, I characterized the adaptor protein Grb2, which is a partner of ErbB2 that is essential for the activation of the MAPK pathway. The solution organization of the domains of this modular protein in its apo-form was unknown so far. I also studied the interaction between Grb2 and CtErbB2, showing that in addition to the known SH2-phosphotyrosine interaction, a polyproline motif of CtErbB2 binds to the N-terminal SH3 domain of Grb2. Finally, I implemented several strategies to phosphorylate CtErbB2 tyrosines, to study more extensively the effect of phosphorylation on the whole tail.

The group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization. / NSERC

La protéine NS5A est essentielle pour la réplication et l'assemblage du virus de l'hépatite C (VHC), et elle constitue une cible thérapeutique prometteuse pour le développement d'antiviraux. Cependant, aucune fonction claire n'a encore été décrite pour NS5A, et les connaissances structurales restent limitées. Ainsi, nous avons caractérisé l'état intrinsèquement désordonné des domaines D2 et D3 de NS5A en décrivant leurs espaces conformationnels et leurs potentialités de repliement en combinant différentes méthodes biophysiques. Nous avons aussi mis en évidence la variabilité structurale du domaine D2 au sein des génotypes du VHC, ce qui pourrait être en rapport avec les différences de pathogénie et d'efficacité des thérapies observées selon les génotypes. L'interaction de D2 et D3 avec la cyclophiline humaine A (CypA) a été étudiée par résonance plasmonique de surface (SPR). Bien que des mutations au sein du domaine D2 rendent la réplication du VHC moins dépendante de la présence de CypA, ces mutations n'empêchent pas la liaison entre D2 et CypA. En revanche, elles induisent des perturbations structurales qui pourraient affecter la cinétique d'interconversion des conformères de D2. Nous avons montré par SPR que D2 et D3 interagissent avec le domaine de fixation à l'ADN du récepteur nucléaire FXR. Cette interaction pourrait inhiber la fixation de FXR sur sa cible ADN, suggérant une implication de NS5A dans la modulation de l'activité transcriptionnelle de ce récepteur nucléaire. L'ensemble de ces informations, nous a permis de proposer un modèle de la structure globale de NS5A permettant une meilleure compréhension des propriétés structurales et fonctionnelles de cette protéine énigmatique / NS5A is essential for HCV replication and particle assembly, and constitutes a very promising drug target. However, no clear function has yet been described for NS5A, and structural knowledge remains limited. We characterized the intrinsically disordered nature of NS5A domains D2 and D3, and describe their folding propensity and their overall conformational behaviour by combining different biophysical methods. We also highlighted the structural variability of D2 domain in HCV genotypes, which might be correlated with the disparities observed between genotypes in terms of pathogenesis and efficiency of therapies. The interactions between D2 and D3 with human cyclophilin A (CypA) was analysed by surface plasmon resonance (SPR). We showed that mutations in the D2 domain conferring resistance of HCV replication to CypA inhibitors did not prevent the interaction between D2 and CypA. However, they induce structural perturbations that may affect the kinetics of conformers interconversion of D2. We also showed by SPR that D2 and D3 interact with the of DNA-binding domain of the nuclear receptor FXR (farnesoid X receptor alpha). This interaction reduce the binding of FXR to its DNA target, suggesting an involvement of NS5A in the modulation of the transcriptional activity of FXR. All this data led us to propose a model of the overall structure of NS5A, which provides a useful template for a better understanding of structural and functional properties of this enigmatic protein

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins are proteins characterized by lack of stable tertiary structures under physiological conditions. Evidence shows that disordered proteins are not only highly involved in protein interactions, but also have the capability to associate with more than one partner. Short disordered protein fragments, called “molecular recognition features” (MoRFs), were hypothesized to facilitate the binding diversity of highly-connected proteins termed “hubs”. MoRFs often couple folding with binding while forming interaction complexes. Two protein disorder mechanisms were proposed to facilitate multiple partner binding and enable hub proteins to bind to multiple partners: 1. One region of disorder could bind to many different partners (one-to-many binding), so the hub protein itself uses disorder for multiple partner binding; and 2. Many different regions of disorder could bind to a single partner (many-to-one binding), so the hub protein is structured but binds to many disordered partners via interaction with disorder. Thousands of MoRF-partner protein complexes were collected from Protein Data Bank in this study, including 321 one-to-many binding examples and 514 many-to-one binding examples. The conformational flexibility of MoRFs was observed at atomic resolution to help the MoRFs to adapt themselves to various binding surfaces of partners or to enable different MoRFs with non-identical sequences to associate with one specific binding pocket. Strikingly, in one-to-many binding, post-translational modification, alternative splicing and partner topology were revealed to play key roles for partner selection of these fuzzy complexes. On the other hand, three distinct binding profiles were identified in the collected many-to-one dataset: similar, intersecting and independent. For the similar binding profile, the distinct MoRFs interact with almost identical binding sites on the same partner. The MoRFs can also interact with a partially the same but partially different binding site, giving the intersecting binding profile. Finally, the MoRFs can interact with completely different binding sites, thus giving the independent binding profile. In conclusion, we suggest that protein disorder with post-translational modifications and alternative splicing are all working together to rewire the protein interaction networks.