Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 144-151).

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 68-71). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (M.S.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

Developmental disorders including: autism, intellectual disability, and congenital abnormalities are present in 3-8% of live births and display a huge amount of phenotypic and genetic heterogeneity. Traditionally, geneticists have identified individual monogenic diseases among these patients but a majority of patients fail to receive a clinical diagnosis. However, the genomes of these patients frequently harbour large copynumber variants (CNVs) but their interpretation remains challenging. Using pathway analysis I found significant functional associations for 329 individual phenotypes and show that 39% of these could explain the patients’ multiple co-morbid phenotypes; and multiple associated genes clustered within individual CNVs. I showed there was significantly more such clustering than expected by chance. In addition, the presence of a multiple functionally-related genes is a significant predictor of CNV pathogenicity beyond the presence of known disease genes and size of the CNV. This clustering of functionally-related genes was part of a broader pattern of functional clusters across the human genome. These genome-wide functional clusters showed tissuespecific expression and some evidence of chromatin-domain level regulation. Furthermore, many genome-wide functional clusters were enriched in segmental duplications making them prone to CNV-causing mutations and were frequently seen disrupted in healthy individuals. However, the majority of the time a pathogenic CNV affected the entire functional cluster, where as benign CNVs tended to affect only one or two genes. I also showed that patients with CNVs affecting the same functional cluster are significantly more phenotypically similar to each other than expected even if their CNVs do not affect any of the same genes. Lastly, I considered one of the major limitations in pathway analysis, namely ascertainment biases in functional information due to the prioritization of genes linked to human disease, and show how the modular nature of gene-networks can be used to identify and prioritize understudied genes.

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. / Protein-protein interactions play a central role in the regulation of biological processes and represent a promissing class of therapeutic targets. It has been recently reported that disrupting the interaction between the PDZ protein PSD-95 and the serotonin receptor 5-HT2A induced an antihyperalgesic effect in diabetic rats. In this context, the development of original ligands capable to inhibit specifically this interaction could lead to a new class of analgesic compounds.We carried out the synthesis of three generations of ligands possessing an indole moiety in order to interact with the highly conserved carboxylate-binding loop (GLGF loop) of PSD-95. Two generations of compounds were developed to find out the position and the nature of the substituents furnishing the best interactions. One generation consists of a family of 15 biligands possessing a substituted indole moiety, coupled with a linker (having from 2 to 6 carbon atoms) via an amid function, ended with various amino acids to interact with the S1 site of the protein, in order to obtain specific ligands.By various biological evaluations, NMR HSQC 1H/15N, chromatography affinity assays and in vivo experiments, we identified two promising inhibitors of the interaction PSD-95/5-HT2A with strong interactions with S0 site of PSD-95. For these compounds, we determined the structure of the complex protein/ligand by NMR NOESY experiments. The orientation of one of these molecules in the S0 site allows us to envisage a new generation of ligands capable to interact with the S1 site of the protein.

abstract: Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in < 1 hour by combining the high throughput and flexible nature of nucleic programmable protein arrays (NAPPA) with the quantitative abilities of surface plasmon resonance imaging (SPRi). NAPPA-SPRi was then used to study the kinetics and affinities of > 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2018

Research of protein-protein interactions produce vast quantities of data and there exists a large number of databases with data from this research. Many of these databases offers the data for download on the web in a number of different formats, many of them XML-based. With the arrival of these XML-based formats, and especially the standardized formats such as PSI-MI, SBML and BioPAX, there is a need for searching in data represented in XML. We wanted to investigate the capabilities of XML query tools when it comes to searching in this data. Due to the large datasets we concentrated on native XML database systems that in addition to search in XML data also offers storage and indexing specially suited for XML documents. A number of queries were tested on data exported from the databases IntAct and Reactome using the XQuery language. There were both simple and advanced queries performed. The simpler queries consisted of queries such as listing information on a specified protein or counting the number of reactions. One central issue with protein-protein interactions is to find pathways, i.e. series of interconnected chemical reactions between proteins. This problem involve graph searches and since we suspected that the complex queries it required would be slow we also developed a C++ program using a graph toolkit. The simpler queries were performed relatively fast. Pathway searches in the native XML databases took long time even for short searches while the C++ program achieved much faster pathway searches.