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F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

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Fungi, diverse and impactful organisms, exert both beneficial and harmful effects on plants, animals, and humans. Certain fungi produce auxin or indole-3-acetic acid (IAA), a crucial plant growth hormone that influences various aspects of plant growth and defense mechanisms. Conversely, pathogenic fungi can produce auxin and manipulate auxin signaling in their host plant to promote fungal virulence and infection progression. Targeting the auxin signaling pathway in pathogenic fungi offers a novel strategy for combating fungal infections in both plants and humans. Nevertheless, the auxin biosynthesis pathway and the role of auxin in fungal symbioses is not fully understood, in part, due to the lack of a tool for measuring intracellular auxin with high spatial and temporal resolution. This dissertation presents the first genetically encoded biosensor engineered from the E3 ubiquitin ligase to detect and quantify intracellular auxin in a Saccharomyces cerevisiae model. The biosensor has been applied to begin studying auxin metabolism and biosynthesis in yeast as well as better understand the plant auxin co-receptor proteins from which it is built. Additionally, the biosensor is re-engineered for application in inducible protein degradation, controlled by auxin. This tool could be applied to identify novel protein targets for disrupting pathogenic fungal species. Overall, this research offers valuable tool and platform for studying auxin biosynthesis pathway, plant protein and auxin signaling as well as intracellular proteins in fungi. / Doctor of Philosophy / Fungi affect plants, animals, and humans, in both beneficial and harmful ways. Some fungi aid other organisms, while others cause illness. Certain fungi produce a hormone called auxin, or indole-3-acetic acid (IAA), which is essential for plant growth and many environmental responses. Auxin can also assist plants in defending against harmful fungi. Conversely, fungi that infect plants can utilize auxin to promote their own growth and spread. Some fungi even produce auxin, possibly aiding in their colonization of plants. In human fungal infection, it is suggested that auxin may be involved in virulent traits and disease progression. Targeting the auxin signaling pathway in harmful fungi presents an innovative approach to combat fungal infections in both plants and humans. However, our understanding of fungal auxin biosynthesis pathways and their role in fungal infections are not fully understood due to the lack of tools to measure auxin in cells efficiently and accurately. This study introduces the first biological tool, called a biosensor, engineered from auxin responsive proteins from plants, to detect and measure intracellular auxin in Baker's yeast. The biosensor has been used to investigate auxin production by yeast. Additionally, the biosensor has been re-engineered for application in inducible protein degradation, controlled by auxin. This tool could be applied to identify novel protein targets for disrupting pathogenic fungal species. Overall, this research provides useful tool and platform to study auxin production, plant protein function and particular proteins in fungi.

Das Ubiquitin-Proteasom-System eukaryotischer Zellen spielt eine zentrale Rolle beim Abbau von fehlgefalteten und nicht mehr benötigten Proteinen. Damit erfüllt es regulatorische Funktionen bei zellulären Prozessen wie z.B. dem Zellzyklus und der Transkription. Das Protein Proteasominhibitor 31 (PI31) wurde als Inhibitor des Proteasoms in vitro charakterisiert. Des weiteren wurde gezeigt, daß überexprimiertes PI31 im murinen System ein Modulator der Assemblierung des Immunoproteasoms (i20S) ist. Über die Funktion und Regulation von PI31 im humanen System war bisher nichts bekannt und wurde deshalb in dieser Arbeit untersucht. Es konnte gezeigt werden, daß neben dem PI31-Transkript mindestens neun weitere alternative Spleißvarianten des humanen PI31 Gens PSMF1 existieren. Die PI31-Isoformen V2 bis V10 unterscheiden sich von PI31 (V1) teils durch eine fehlende N-terminale Domäne oder einen veränderten C-Terminus. Die Isoform V5 wird als einzige gewebespezifisch in Testikeln exprimiert und ist im Zellkern lokalisiert. Ausschließlich die Überexpression der Isoform V3 führt zur Inhibition der proteasomalen Aktivität in vivo. Ein modulatorischer Einfluß von PI31 oder einer der Isoformen auf die Assemblierung des humanen i20S bestätigte sich dagegen nicht. Die Überexpression von PI31 und V3 in humanen Zellen führte indes zu einer Akkumulation und verzögerten Degradation von proteasomalen Substraten. Es wurde außerdem gezeigt, daß die Expression von humanem PI31 durch virusassoziierte Stimuli wie dsRNA und Typ I-Interferone induziert werden kann. Für die 3kb lange 3’UTR der PI31-mRNA konnte zusätzlich nachgewiesen werden, daß sie inhibitorisch auf die Expression wirkt und somit eine regulatorische Funktion besitzt. In Zusammenhang mit der von Kirk et al. (2008) gezeigten Heterodimerisierung von PI31 mit dem F-Box Protein Fbxo7, weisen die hier vorgestellten Ergebnisse auf eine Funktion von PI31 und dessen Isoformen bei der Ubiquitinierung von proteasomalen Substraten hin. / The ubiquitin–proteasome pathway is the major intracellular system for protein degradation. It plays an important role in the regulation of cellular processes like cell cycle control, signal transduction and gene transcription. The protein proteasome inhibitor 31 (PI31) was initially characterized as a potent inhibitor of proteasomal activity in vitro. Furthermore it was shown that PI31 modulates the assembly of the murine immunoproteasome (i20S). The function and regulation of PI31 in the human system is so far unexplored and therefore the topic of this study. It was shown that at least nine alternatively spliced variants of the PI31 gene PSMF1 exist additionally to the PI31 transcript. The PI31 isoforms V2 to V10 differ from PI31 (V1) in parts of the N-terminus and in a modified C-terminus. Only the isoform V5 is tissue specific expressed in testis and localized in the nucleus. After overexpression only the isoform V3 has the ability to inhibit the proteasomal activity in vivo. In contrast to the murine system neither PI31 nor the isoforms showed a modulatory effect on the assembly of the i20S. The overexpression of PI31 and V3 in human cells results instead in the accumulation and delayed degradation of proteasomal substrates. Furthermore the expression of human PI31 can be induced by virus associated stimuli like dsRNA and type I interferones. In addition, for the 3kb long 3’UTR of the PI31-mRNA an inhibitory effect on the expression and therefore a regulatory role was shown. Together with data from Kirk et al. (2008), who show the heterodimerization of PI31 with the F-box protein Fbxo7, the presented results suggest a function of PI31 and its isoforms in the process of ubiquitination of proteasomal substrates.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-104).

Barley and wheat are the two most important crop species in Turkey. Molecular studies for increasing crop yield of these species are very important for the economic benefits of Turkey. Powdery mildew and yellow rust are the two main pathogens, infecting barley and wheat, respectively in our country and causing a great amount of yield loss each year. Till now, classical genetics studies were performed in order to develop resistant barley and wheat cultivars, but these studies have not been succesful. Therefore, molecular plant-pathogen interactions studies are starting to become the new tool to fight against pathogens. In this thesis, two important aspects of plant microbe interactions were investigated. In the first part, the role of microRNAs (miRNAs) in powdery mildew-barley pathosysytem, and yellow rust-wheat pathosystem were studied. The expression levels of miRNAs and their putative targets were investigated via miRNA microarray analysis and qRT-PCR, respectively, in response to virulent and avirulent pathogen infections. These data were used to establish a new model for powdery mildew-barley and yellow rust-wheat pathosystems. In the second part, functional analysis of a novel F-box gene, which was a ZTL-type F-box, was performed by using Barley Stripe Mosaic Virus mediated Virus Induced Gene Silencing. This F-box gene (HvDRF) (Hordeum vulgare Disease Related F-box) was induced upon yellow rust infection and we studied its role in powdery mildew infection. The results confirmed HvDRF as a positive regulator of race specific immunity and enlarged the roles of ZTL-type F-box proteins to biotic stress responses.

Les modifications post-traductionnelles des protéines constituent un niveau crucial de régulation de l’expression des gènes. Parmi elles, la conjugaison peptidique impliquant l’ubiquitine intervient entre autre dans la régulation de la stabilité protéique. La fixation de ce peptide de 76 acides aminés, extrêmement conservé, sous forme de chaîne de polyubiquitine, nécessite l’intervention de trois enzymes (E1, E2 et E3) et constitue un signal de dégradation de la protéine ainsi modifiée. Cette voie de régulation intervient dans de très nombreux processus biologiques. Les complexes SCF sont impliqués dans la voie de protéolyse ciblée. Ils représentent l' une des classes les plus fréquentes d'ubiquitine ligase E3 et ils sont composés de quatre sous-unités (Rbx, Cullin, SKP1, et F-box). La structure et la fonction des complexes SCF, ont été étudiées chez la levure, l’Homme et la plante modèle A. thaliana. Cependant, peu de travaux ont été réalisés chez des plantes cultivées, en particulier les céréales, telles que le blé. Cinq gènes codant pour la sous-unité Skp1 (TSK1, TSK3, TSK6, TSK11 et TSK16), cinq gènes codant pour la sous-unité F-box (ZTL, ATFBL5, EBF, TIR1 et ABA-T), un gène codant pour la sous-unité Cullin1 et un gène codant pour la protéine RBX du complexe SCF du blé, ont été isolés et clonés. Les différents tests d’interaction entre les quatre sous-unités du complexe SCF ont été réalisés par la méthode du double-hybride dans la levure en utilisant la technologie Gateway. Ces études ont montré que les deux protéines, TSK1 et TSK3, fixent spécifiquement différentes sous-unités F-box. Parallèlement, nous avons montré que la protéine TSK11 représente une structure particulière. Des études d’insertion/délétion sur la protéine TSK11 ont permis d’identifier un nouveau domaine indispensable à l’interaction. Les analyses par PCR semi-quantitative des différents gènes codant pour la sous-unité Skp1, dans trois tissus différents (feuille tige et racine), ont mis en évidence une expression constitutive des gènes TSK3, TSK6 et TSK11. Tandis que les gènes TSK1 et TSK16 sont exprimés préférentiellement dans les racines. Les analyses par PCR semi-quantitative sur des plantules de blé à différents stades de développement, ont mis en évidence une surexpression du gène TSK11 au moment de la floraison. Ce qui suggère que TSK11 est probablement un équivalent fonctionnel d’ASK1 chez Arabidopsis thaliana. / The selective degradation of proteins is an important means of regulating gene expression and plays crucial roles in the control of various cellular processes. The Ubiquitin (Ub)–Proteasome System (UPS) is the principal non-lysosomal proteolytic pathway in eukaryotic cells and is required for the degradation of key regulatory proteins. Ubiquitin is a 76-residue protein that can be attached covalently to target proteins through an enzymatic conjugation cascade involving three enzymes denoted, E1, E2 and E3.The SCF complex is a type of ubiquitin-protein ligase (E3) that acts as the specific factor responsible for substrate recognition and ubiquitination. Some polyubiquitinated proteins are then targeted to the 26S proteasome for degradation. The SCF complex consists of four components including SKP1, Cullin1, Rbx1 and a large gene family of F-box proteins. Twenty one SKP1-related genes have been described in the Arabidopsis genome and some of these genes have been analyzed genetically. By contrast, little is known about the function and structure of SKP1 homologues in wheat. Some of the Triticum SKP1-related protein (TSKs) have been characterized in this study. Five complete sequences of SKP1 (TSK1, TSK3, TSK6, TSK11 and TSK16), five F-box (ZTL, ATFBL5, EBF, TIR1 and ABA-T), one Cullin1 and one Rbx, were successfully cloned and biochemically characterized. Yeast two-hybrid analysis showed that TSK1 and TSK3 are capable of interacting with different F-box proteins. Furthermore, TSK11 contains an additional domain that changed its interaction capabilities. In vitro analysis using a chimeric protein showed that this additional domain could modify the interaction between a SKP-like protein and two F-box proteins. Expression analyses revealed that TSK1 and TSK16 were expressed predominantly in roots. While, TSK3, TSK6 and TSK11 were expressed in several wheat organs. In addition, the TSK11 was up-regulated in the leaves at the flowering stage.

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