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

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

L'ubiquitylation des protéines est une modification post-traductionnelle qui joue un rôle capital dans la régulation des nombreuses fonctions cellulaires, y compris la croissance cellulaire et la prolifération. Les dysfonctionnements de ce mécanisme sont à l'origine de diverses maladies telles que le cancer par exemple. Le processus d'ubiquitylation implique une série des réactions enzymatiques en cascade, catalysées par une famille des enzymes, structuralement très proches. Cette famille est composée des enzymes activateurs d'ubiquitine (E1s), des enzymes de conjugaison d'ubiquitine (E2s) et des ligases d'ubiquitine (E3s). Les interactions entre E2s et E3s sont dans le centre de la cascade d'ubiquitylation. Une combinaison particulière des pairs E2/E3 va déterminer le type de chaînes d'ubiquitine qui seront attachées à la protéine d'intérêt pour ensuite déterminer la fonction régulatrice de la voie d'ubiquitylation. A ce jour, seulement une petite fraction de paires possibles entre E2 et E3 a été investiguée par des approches biochimiques et in vitro. Cependant ces approches ne reflètent pas forcément des conditions qu'on trouve dans une cellule vivante. Prenant ceci en considération, les principales objectives de ma thèse seront comme suit : identifier et optimiser une méthode de détection et de quantification des interactions E2/E3 dans une cellule vivante de la levure de boulanger (Saccharomyces cerevisiae) ; construire une bibliothèque de souches de la levure qui permettrait d'établir des interactions entre E2 et E3 ; chercher de nouvelles potentielles paires E2/E3 ; caractériser fonctionnellement une potentielle paire E2/E2. Il est difficile de trouver une méthodologie appropriée afin d'étudier les interactions entre E2 et E3 parce qu'ils sont relativement faibles et transitoires. Leurs études nécessitent donc des techniques de détection avec une grande sensibilité. Parmi différentes techniques nous avons testé et choisi la complémentation bimoléculaire de la fluorescence, BiFC. Kurtosis, une mesure permettant localiser et quantifier la fluorescence BiFC-spécifique. Nos résultats nous nous avons permis à identifier 117 putatives paires E2/E3 parmi quels, 23 paires ont été déjà décrit dans la littérature. Parmi 94 nouvelles paires, certains E3s interagissent avec seulement une seule E2 ou d'autres donnent un signal BiFC avec plusieurs E2s. Ubc13, Ubc1 et Ubc4 sont les E2s qui interagissent le plus souvent. Nous avons identifié aussi une interaction entre les protéines Asi1 et Asi3 et les enzymes de conjugaison d'ubiquitine Ubc6 et Ubc7. Asi1 et 3 sont connus de former un complexe Asi1/3 sur la membrane intérieure du noyau impliqué dans la réponse de la cellule aux acides aminés extracellulaires. Ces protéines contiennent un domaine RING caractéristique pour les ligases d'ubiquitine mais cette activité n'était pas démontrée auparavant. / Protein ubiquitylation is a post-translational modification that plays a crucial role in regulating many cellular functions, including cell growth and proliferation. Defects in this control mechanism cause cancer and other diseases. The ubiquitylation process involves a cascade of enzymatic reactions catalyzed by a family of structurally-related enzymes, namely ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s). Interactions between E2s and E3s are in the centre of ubiquitylation cascade and it is a combination of particular E2/E3 pairs that determine what types of ubiquitin chains are made, thus determining the regulatory functions of the ubiquitin pathway. To date, only a small fraction of all possible E2/E3 pairs have been investigated, mainly using biochemical and in vitro approaches that may not accurately reflect the conditions that occur in living cells. We aimed to develop a method capable of detecting specific E2-E3 interactions under physiological conditions. Using budding yeast as a model organism, we found that the Bimolecular Fluorescence Complementation (BiFC) enables sensitive detection of the well described Ubc4-Ufd4 pair under endogenous conditions. The assay is specific since the interaction signal is lost in yeasts expressing Ubc4 mutants truncated in its E3 interaction domain. We then used this system to further analyze the physiological network of E2 and E3 enzymes in living yeast. We performed a microscopy screen to assay all interactions between eleven E2s and 56 E3s. Our results show that approximately 20% of all E2/E3 combinations give a detectable BiFC signal. Few E3s interacted only with a single E2, whereas most E3s produced a BiFC signal with multiple E2s. Ubc13, Ubc1 and Ubc4 were found to be the most frequently interacting E2s. Our results match many examples from current literature but we also detected 94 new E2/E3 interactions, in particular we identified an interaction between the proteins Asi1 and Asi3 and E2s Ubc6 and Ubc7. Asi1 and Asi3 are known to form a complex (the Asi1/3 complex) at the inner nuclear membrane and are involved in the regulation of the response to extracellular amino acids. The Asi1/3 complex was suspected to function as a ubiquitin ligases because they contain a RING domain, but this has previously not been demonstrated. We therefore further characterized them functionally.

Activation of otherwise unreactive substrates is a common strategy in chemistry and in nature. Adenylate-forming enzymes use adenosine monophosphate to activate the hydroxyl of their carboxylic substrate, creating a better leaving group. In a second step this reactive group is replaced in a nucleophilic elimination reaction to form esters, amides or thioesters. Recent studies have revealed that NRPS- independent siderophore (NIS) synthetases are also adenylate-forming enzymes, but are not included in the current superfamily description. NIS enzymes are involved in biosynthesis of high-affinity iron chelators which are used for iron acquisition by many pathogenic microorganisms. This is an important area of study, not only for potential therapeutic intervention, but also to illuminate new enzyme chemistries. Here the structural and biochemical studies of AcsD from Pectobacterium chrysanthemi are reported. AcsD is a NIS synthetase involved in achromobactin biosynthesis. The co-complex structures of ATP and citrate provide a mechanism for the stereospecific formation of an enzyme-bound citryl-adenylate. This intermediate reacts with L-serine to form a likely achromobactin precursor. A detailed characterization of AcsD nucleophile profile showed that it can not only catalyze ester formation, but also amide and possibly thioester formation, creating new stereospecific citric acid derivatives. The structure of a N-citryl-ethylenediamine product co-complex identifies the residues that are important for both recognition of L-serine and for catalyzing ester formation. The structural studies on the processive enzyme AlcC, which is involved in the final step of alcaligin biosynthesis of Bordetella pertussis, show that it has a similar topology to AcsD. It also shows that ATP is coordinated in a manner similar to that seen in AcsD. Biochemical studies of a substrate analogue establish that AlcC is not only capable of synthesizing substrate dimers and trimers, but also able to assemble the respective dimer and trimer macrocycles. A series of docked binding models have been developed to illustrate the likely substrate coordination and the steps along dimerization and macrocyclization formation. Structural and mechanistic comparison of NIS enzymes with other adenylate-forming enzymes highlights the diversity of the fold, active site architecture, and metal coordination that has evolved. Hence, a new classification scheme for adenylate forming enzymes is proposed.

Previously it has been shown that Rsp5p, a member of Nedd4 ubiquitin ligases in yeast, is modified by the ubiquitin-like protein SUMO and that this modification is performed by Siz1p, a member of PIAS SUMO ligases that are in turn substrates of Rsp5p-dependent ubiquitylation, thus defining a previously unidentified system of crosstalk between the ubiquitin and SUMO systems in yeast. This project aims to identify whether similar crosstalk pattern exists in human cells. In vitro ubiquitylation assays showed that some of the human Nedd4 family members (Nedd4.1, Nedd4.2, WWP1) are capable of ubiquitylating the human SUMO ligase PIAS3, while in contrast, Smurf2 does not appear to be able to modify this protein. This modification is partially WW-PY-motif-dependent as ubiquitylation level of PIAS3 mutants with altered PY motifs conducted by Nedd4.1 or Nedd4.2 was reduced, but not completely disrupted. Interestingly, in vitro SUMOylation assay revealed that Nedd4.1 is SUMOylated even in the absence of SUMO E3 ligases and an apparent interaction between the SUMO E2 (Ubc9) and Nedd4.1 was observed both in vitro and in vivo. I show that auto- SUMOylation of Nedd4.1 is accompanied with the formation of thioester-linked conjugates between Nedd4.1 and SUMO, but these do not involve cysteine residues (C867, C778, and C627) within the HECT domain itself and is not occurring at a predicted SUMOylation consensus site (K357). Furthermore, I have shown that Nedd4.1 and SUMO1/2 colocalize in HeLa cells, and that overexpression of epitope tagged Nedd4 and SUMO1/2, followed by denaturing pull-downs demonstrates that both Nedd4.1 and Nedd4.2 can be SUMOylated in vivo. Meanwhile, I have generated a SUMO trap based on SUMO interacting motifs (SIMs) and confirmed its ability of capturing SUMOylated proteins both in vivo and in vitro. Its use reveals that Nedd4 SUMO conjugates could be captured by SUMO trap when Nedd4 and SUMO were co-expressed in HeLa cells, again confirming Nedd4.1 as a substrate for SUMO1 or SUMO2. In conclusion, I show that SUMOylation of Nedd4.1 does exist in HeLa cells, and on the other hand, some of Nedd4 family members are responsible for PIAS3 ubiquitylation in vitro, providing evidence of a crosstalk between Nedd4 family of ubiquitin ligases and PIAS family of SUMO ligases in mammals.