L'analyse cinétique d'interactions de protéines offre une multitude d'informations sur les fonctions physiologiques de ces molécules au sein de l'activité cellulaire, et peut donc contribuer à l'amélioration des diagnostics médicaux ainsi qu'à la découverte de nouveaux traitements thérapeutiques. La résonance plasmonique de surface (SPR) est la technique de biodétection optique de référence pour les études cinétiques d'interaction de molécules biologiques. Si la SPR offre une détection en temps réel et sans marquage, elle nécessite en revanche des équipements coûteux et sophistiqués ainsi que du personnel qualifié, limitant ainsi son utilisation au sein de laboratoires de recherche académiques. Dans ces travaux de thèse, nous avons développé une plateforme de biodétection basée sur l'utilisation de nanofentes biofonctionnalisées combinées avec une détection par microscopie à fluorescence. Ce système permet l'observation en temps réel d'interactions protéines-protéines et la détermination des constantes cinétiques associées, avec des temps de réponse optimisés et une excellente efficacité de capture. La fonctionnalité du système a été démontrée par l'étude des cinétiques d'interaction de deux couples modèles de différentes affinités : le couple streptavidine/biotine et le couple IgG de souris/anti-IgG de souris. Une très bonne cohérence entre les constantes cinétiques extraites, celles obtenues par des expériences similaires réalisées en SPR et les valeurs rapportées dans la littérature montre que notre approche pourrait être facilement applicable pour l'étude cinétique d'interactions de protéines avec une sensibilité allant jusqu'au pM, sur une large gamme de constantes de dissociation. De plus, nous avons intégré un générateur de gradient de concentrations microfluidique en amont de nos nanofentes, permettant ainsi des mesures simultanées de cinétiques d'interactions à différentes concentrations d'analyte en une seule expérience. Ce système intégré offre de nombreux avantages, tels qu'une réduction de la consommation des réactifs et des temps d'analyse par rapport aux approches séquentielles classiques. Cette technologie innovante pourrait ainsi être un outil précieux non seulement pour les domaines du biomédical et de la médecine personnalisée mais aussi pour la recherche fondamentale en chimie et biologie. / Kinetic monitoring of protein-protein interactions offers fundamental insights of their cellular functions and is a vital key for the improvement of diagnostic tests as well as the discovery of novel therapeutic drugs. Surface plasmon resonance (SPR) is an established biosensor technology routinely used for kinetic studies of biomolecular interactions. While SPR offers the benefits of real-time and label-free detection, it requires expensive and sophisticated optical apparatus and highly trained personnel, thus limiting the accessibility of standard laboratories. In this PhD project, we have developed an alternative and cost-effective biosensor platform exploiting biofunctionalized nanofluidic slits, or nanoslits, combined with a bench-top fluorescence microscope. Our approach enables the visualization of protein interactions in real-time with the possibility to determine associated kinetic parameters along with optimized response times and enhanced binding efficiency. We have demonstrated the effectiveness of our devices through kinetic studies of two representative protein-receptor pairs with different binding affinities: streptavidin-biotin and mouse IgG/anti-mouse IgG interactions. Good agreement of extracted kinetic parameters between our device, SPR measurements and literature values indicated that this approach could be readily applicable to study kinetics of protein interactions with sensitivity down to 1 pM on a large scale of dissociation constants. In addition, we have incorporated a microfluidic gradient generator to our validated nanoslit device, which has allowed one-shot parallel kinetic measurements to be realized in a single-experiment. This integrated system provides advantages of diminished material consumption and analysis time over the conventional kinetic assays. We believe that this innovative technology will drive future advancements not only in the discipline of biomedical and personalized medicine, but also in basic chemical/biological research.

MSc (Biochemistry) / Department of Biochemistry / Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support ii the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte. / NRF

It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.

La méiose est un processus de ségrégation des chromosomes essentiel pour la gamétogenèse chez tous les organismes qui présentent une reproduction sexuée. Ce mécanisme nécessite des connections entre chromosomes homologues et des structures intermédiaires d’ADN appelées Jonction de Holliday. Ces jonctions sont résolues principalement par complexe MutLγ (Mlh1-Mlh3). Des mutations sur les gènes impliqués en méiose s’accompagnent chez l’homme de problèmes allant de la stérilité à des réarrangements chromosomiques comme la trisomie. Mlh1-Mlh3 joue aussi un rôle dans la voie de réparation des mésappariements de l’ADN (MisMatch Repair - MMR). Notre laboratoire a révélé la première structure cristalline de la région C-terminale du complexe MutLα (Mlh1-Pms1) qui est l’endonucléase majeure de la voie MMR. Mon projet de thèse s’insère dans le cadre de l’étude des différentes fonctions des MutL eucaryotes et plus particulièrement sur le mécanisme moléculaire de MutLγ (Mlh1-Mlh3). Au cours de ma thèse, nous avons déterminé la structure cristalline du domaine C-terminale du complexe Mlh1-Mlh3 qui contient le site d’endonucléase et caractérisé 3 états du site actif. Nous avons montré le rôle de Mlh1 dans le site endonucléase. Nous avons caractérisé la spécificité de ce domaine pour les Jonctions de Holliday et proposé un modèle du site de fixation de l’ADN sur le complexe entier. Ce modèle a permis de proposer un nouveau mutant de séparation de fonction de Mlh1-Mlh3, appelé KERE, qui a été analysé par gel retard et génétique. / Meiosis is key process in sexual reproduction, where chromosomes are segregated. During this process, a parental diploid cell divides into haploid gametes. This mechanism requires connections between homologous chromosomes and intermediate DNA structures called Holliday Junctions. These junctions are mainly resolved by MutLγ (Mlh1-Mlh3) complex. Mutations of genes involved in meiosis are associated with human diseases including sterility and chromosomal rearrangements such as trisomy. Mlh1-Mlh3 plays also a role in DNA mismatch repair (MMR). Our laboratory has characterized the first crystal structure of the C-terminal region of the MutLα complex (Mlh1-Pms1) which is the major endonuclease in MMR. My thesis aims at understanding the molecular mechanism of MutLγ (Mlh1-Mlh3) mainly involved in meiosis and to compare it with Mlh1-Pms1 mainly involved in MMR.We determined the crystal structure of the C-terminal domain of the MutLγ complex which contains the endonuclease site. We characterized the structure of three different states of the active site. We showed how Mlh1 is an integral part of the Mlh3 endonuclease site. We characterized the specificity of this domain for Holliday Junctions and proposed a model of the full-length complex and its DNA binding sites. Finally, we design new separation of function allele of Mlh1-Mlh3, called KERE, which was analyzed by EMSA and genetic experiments.

La résistance acquise aux traitements anticancéreux représente un problème clinique majeur. Les voies de réparation de l'ADN fournissent un mécanisme de résistance, mais celle-ci peut aussi résulter de mutations ou d'une expression réduite de la protéine ciblée. La surexpression ou la localisation nucléaire de la Y-Box binding (YB-1) protéine est considérée comme un marqueur pronostique de chimiorésistance de la tumeur. YB-1 interagit avec plusieurs partenaires ; dans cette étude, nous nous sommes concentrés sur son interaction avec l'enzyme de réparation de l'ADN NTH1 (hNTH1) et l'ADN topoisomérase I (hTopoI), deux enzymes stimulées par YB-1. L'abondance du complexe hNTH1/YB-1 est accrue dans les cellules tumorales résistantes au cisplatine. La TopoI humaine est une enzyme essentielle impliquée dans la régulation cellulaire du surenroulement de l'ADN et est la cible de plusieurs agents anticancéreux. YB-1 augmente la sensibilité à l'inhibiteur de TopoI, la camptothécine, dans les tumeurs. Nous avons caractérisé les complexes YB-1/hNTH1 et YB-1/hTopoI in vitro et in vivo en utilisant des mesures de transfert d'énergie par résonance en fluorescence (ou FRET) pour identifier et développer de nouvelles stratégies pour le traitement de tumeurs chimio-résistantes. Nous avons développé et optimisé un biosenseur original basé sur le FRET pour cribler deux chimiothèques de taille moyenne afin d’identifier des inhibiteurs potentiels du complexe hNTH1/YB-1. Plusieurs «hits» ont été identifiés qui réduisent de façon significative le niveau de FRET de notre biosenseur. Pour certains de ces composés, nous avons reproduit ces résultats à partir de poudres, effectué des courbes dose-réponse et validé leurs actions en tant qu'inhibiteurs de l'interface hNTH1/YB-1 en utilisant d'autres tests d’interactions. Ensemble nos résultats démontrent que YB-1 interagit directement et stimule des enzymes de la réparation de l'ADN et du relaxation de l’ADN, et que cibler l’interface YB-1/hNTH1 représente une nouvelle stratégie intéressante pour le développement de traitements anticancéreux. / Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several “hits” were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs.

Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.

Indiana University-Purdue University Indianapolis (IUPUI) / Non-alcoholic fatty liver disease and metabolic syndrome induced by high nutrient status have increasingly become a global health concern as it cause multiple complications. The mTOR complex is central in regulating anabolic reactions within cells under growth factors or under high nutrients stimulation. Constitutive and persistent activation of mTOR can impair cellular functions. In the first part of this study, we demonstrate a damping oscillation of mTOR activity during a long-term treatment of high fat diet. TFEB translocation and lysosomal enzyme activity also oscillate, but in an opposite direction. TFEB controls the lysosomal activity, autophagic degradation and lipid metabolism. Overexpression of wild type and mutant TFEB could inhibit NAFLD development in mice. In addition, TFEB location in nucleus inversely correlates with NAFLD severity in patients. mTOR activation under hypernutrition status suppresses TFEB translocation, inhibits lysosomal functions and autophagic degradation of lipid droplets. Inhibition of mTOR activity by rapamycin reverse the above phenotypes. Because mTOR activation also requires normal lysosomal function, the inhibition of TFEB by mTOR leads to decreased lysosomal function and mTOR downregulation. This negative feedback may explain the oscillation pattern of mTOR activation in long term high fat diet regimen and is a novel mechanism for inhibition of mTOR. In the second part of study, we report that Bid protein, previously known for its pro-apoptosis function in promoting mitochondrial permeability, plays an unexpected role in regulating fatty acid beta oxidation. Deletion of Bid in mice reprograms the body's response to hyper-nutrition caused by high fat diet, leading to the resistance to the development of obesity, liver steatosis and metabolic syndrome. These mice present a higher oxygen consumption, a lower respiratory quotient, and an increased beta-oxidation rate. Mechanistically, the high fat diet regimen triggers translocation of the full length Bid molecule to mitochondrial membrane. Genetic deletion of Bid also affects the stability of its binding protein, MTCH2 in the mitochondrial membrane. In summary, we describe in this study a mTOR-TFEB-lysosome feedback loop, which can regulate NAFLD development, and a novel Bid-mediated regulatory mechanism in beta-oxidation, which limits energy expenditure and promotes obesity development.

No description available.

BACKGROUND: A long-standing challenge in the post-genomic era of Bioinformatics is the prediction of protein-protein interactions, and ultimately the prediction of protein functions. The problem is intrinsically harder, when only amino acid sequences are available, but a solution is more universally applicable. So far, the problem of uncovering protein-protein interactions has been addressed in a variety of ways, both experimentally and computationally. MOTIVATION: The central problem is: How can protein complexes with solved threedimensional structure be utilized to identify and classify protein binding sites and how can knowledge be inferred from this classification such that protein interactions can be predicted for proteins without solved structure? The underlying hypothesis is that protein binding sites are often restricted to a small number of residues, which additionally often are well-conserved in order to maintain an interaction. Therefore, the signal-to-noise ratio in binding sites is expected to be higher than in other parts of the surface. This enables binding site detection in unknown proteins, when homology based annotation transfer fails. APPROACH: The problem is addressed by first investigating how geometrical aspects of domain-domain associations can lead to a rigorous structural classification of the multitude of protein interface types. The interface types are explored with respect to two aspects: First, how do interface types with one-sided homology reveal convergently evolved motifs? Second, how can sequential descriptors for local structural features be derived from the interface type classification? Then, the use of sequential representations for binding sites in order to predict protein interactions is investigated. The underlying algorithms are based on machine learning techniques, in particular Hidden Markov Models. RESULTS: This work includes a novel approach to a comprehensive geometrical classification of domain interfaces. Alternative structural domain associations are found for 40% of all family-family interactions. Evaluation of the classification algorithm on a hand-curated set of interfaces yielded a precision of 83% and a recall of 95%. For the first time, a systematic screen of convergently evolved motifs in 102.000 protein-protein interactions with structural information is derived. With respect to this dataset, all cases related to viral mimicry of human interface bindings are identified. Finally, a library of 740 motif descriptors for binding site recognition - encoded as Hidden Markov Models - is generated and cross-validated. Tests for the significance of motifs are provided. The usefulness of descriptors for protein-ligand binding sites is demonstrated for the case of "ATP-binding", where a precision of 89% is achieved, thus outperforming comparable motifs from PROSITE. In particular, a novel descriptor for a P-loop variant has been used to identify ATP-binding sites in 60 protein sequences that have not been annotated before by existing motif databases.

No description available.