Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Pharmaceutical Science." Includes bibliographical references (p. 99-107).

Human ISG15 is a 17 kDa ubiquitin-like protein (Ubl) that is induced by type I interferons (interferons [alpha] and [beta]) and plays a role in antiviral responses. ISG15 is conjugated via its C-terminus to more than 150 cellular proteins, and like ubiquitin, an E1-E2-E3 enzymatic cascade is required for conjugation. Ube1L and UbcH8 were previously identified as the E1 and E2 enzymes for this pathway. My experiments identified Herc5, a HECT domain E3, as the major ligase for ISG15. Like ISG15, Ube1L, and UbcH8, expression of Herc5 is transcriptionally induced by type I interferons. siRNAs against Herc5 abrogated ISG15 conjugation to the vast majority of target proteins in interferon-treated cells. Wild type Herc5, but not the catalytically inactive C994A mutant, supported conjugation of ISG15 in non-interferon-treated cells co-transfected with Ube1L, UbcH8 and ISG15. IQGAP1, a scaffold protein, was identified as another essential component of the ISG15 system. IQGAP1 was discovered to interact with Herc5, and this interaction was mediated by the C-terminal domain of IQGAP1 and the N-terminal RCC1-like repeats of Herc5. IQGAP1 was required for auto-conjugation of ISG15 to Herc5, and I propose a model where IQGAP1 functions, at least in part, by relieving an auto-inhibitory conformation of Herc5. Thus, I have identified two factors that are critical for ISG15 conjugation and my discoveries have increased our understanding of the ISG15 pathway. Identification and characterization of the conjugation apparatus will aid in establishing an in vitro biochemical system for ISG15 conjugation, which in turn, will be important to decipher the biological function of ISG15 modification. / text

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 105-116). Also available on the World Wide Web.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/08). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 177-200). Also available on the Internet.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 178-184).

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 159-179).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Human glutathione synthetase (hGS) is a homodimeric enzymes that catalyzes the second step in the biological synthesis of glutathione, a critical cellular antioxidant. The enzyme exhibits negative cooperativity towards the γ-glutamylcysteine (γ-GC) substrate. In this type of allosteric regulation, the binding of γ-GC at one active site significantly reduces substrate affinity at a second active site over 40 Å away. The presented work explores protein-protein interactions, substrate binding, and allosteric communication through investigation of three regions of hGS: the dimer interface, the S-loop, and the E-loop. Strong electrostatic interactions across the dimer interface of hGS maintain the appropriate tertiary and quaternary enzymatic structure needed for activity. The S-loop and E-loop of hGS form walls of the active site near γ-GC, with some residues serving to bind and position the negatively cooperative substrate. These strong interactions in the active site serve as a trigger for allosteric communication, which then passes through hydrophobic interactions at the interface. A comprehensive computational and experimental approach relates hGS structure with activity and regulation. ATP-grasp enzymes, including hGS, utilize ATP in the nucleophilic attack of a carboxylic acid in a reaction thought to proceed through the formation of an acylphosphate intermediate. Small metal cations are known to chelate the terminal phosphates of actives site ATP, yet the role of these atoms remains unclear. In the presented work, a computational metal substitution study establishes the role these divalent cations in the catalysis of peptide bonds. The simple model is used to determine the impact of metal cations on the thermodynamics and kinetics, an important stepping stone in understanding the importance of metal cations in larger biological systems.

Les courts ARN non codants tels que les microARN, les piARN et les siARN sont de petites molécules d’ARN de 20 à 30 nucléotides de long qui sont très bien conservées au cours de l’évolution. Elles s’associent à des protéines Argonautes afin de former un complexe effecteur appelé RISC (RNA induced silencing complex). Ces courtes séquences, ne codant pour aucune protéine, agissent comme de puissants régulateurs de l’expression des gènes. De nombreuses évidences supportent qu’une dérégulation du niveau d’expression de ces courts ARN non codants contribue au développement et au maintien de nombreuses pathologies telles que le cancer. De ce fait, il est essentiel pour la cellule de contrôler la stabilité des courts ARN non codants. Le contrôle de la maturation et de la stabilité de ces courts ARN non codants sont des mécanismes peu connus. L’objectif principal de mon doctorat a donc été de mieux comprendre comment le niveau des courts ARN non codants est contrôlé. Afin d’étudier plus en détail comment le niveau des microARN est régulé, nous avons identifié la phosphatase PPM-2 (PP2Cα chez l’humain) et l’E3 ubiquitine ligase HECD-1 (HectD1 chez l’humain) comme étant de nouveaux interacteurs du complexe de dégradation des microARN. Nous avons utilisé des approches de génétique et de biologie moléculaire chez le nématode C. elegans, pour étudier le rôle de la perte de fonction de ppm-2 et d’hecd1 dans la voie des courts ARN non codants. Nos travaux ont montré que la perte de fonction de ppm-2 induit des défauts développementaux qui sont associés à des défauts de la voie des microARN. De plus, l’absence de ppm-2 exacerbe les phénotypes développementaux observés dans des animaux où la voie des microARN est altérée. De manière intéressante, chez le mutant ppm-2, nous avons constaté que d’autres voies de courts ARN non codants, telles que la voie des piARN et celle de l’endosiARN nucléaire, sont affectées. Du point de vue moléculaire, nous avons observé une déstabilisation du niveau d’expression de plusieurs protéines Argonautes dans le mutant ppm-2. En effet, ces dernières sont envoyées à la dégradation par la voie du protéasome seulement chez des animaux mutés pour ppm-2. Concernant l’étude de HECD1, nous avons remarqué que la perte de fonction de cette ubiquitine ligase entrainait une diminution de la progéniture et une létalité embryonnaire attribuable à des défauts dans la gamétogénèse. De plus, nous avons observé une accumulation de miARN fonctionnels chez des animaux mutés pour hecd-1. L’ubiquitine ligase HECD-1 pourrait être impliquée dans la transcription ou la dégradation des miARN. En conclusion, nos résultats suggèrent que PPM-2 permet de contrôler la stabilité des protéines Argonautes en les dirigeant dans une voie alternative de dégradation et que l’ubiquitine ligase HECD-1 pourrait être impliquée dans la régulation des miARN en modulant leur transcription ou leur dégradation. Mes travaux de doctorat nous ont permis de mettre en lumière un nouveau modulateur des courts ARN non codants, PPM-2, qui agit via le contrôle de la régulation des Argonautes. Les avancées de la recherche dans le domaine des courts ARN non codants pourra permettre le développement de nouvelles thérapies. / Small non-coding RNAs, like microRNAs, piRNAs or siRNAs, are small RNA molecules, 20 to 30 nucleotides long that are conserved during evolution. They form an induced silencing complex (RISC) in association with Argonaute proteins to regulate gene expression. Small non-coding RNAs are involved in the regulation of genes implicated in cell proliferation, differentiation and development. Many evidences support that deregulation of the expression level of those small non-coding RNAs contribute to the development of pathologies such as cancer. It is therefore essential for cells to control small non-coding RNA stability. The control of maturation and stability of those small molecules are poorly understood. The main objective of my doctorate was to better understand how the stability of small non-coding RNAs is controlled. In order to study in more detail how miRNAs are regulated, we identified two factors involved in miRNA turnover in C. elegans. We found that the phosphatase PPM-2 (PP2Cα in human) and the E3 ubiquitin ligase HECD-1 (HectD1 in human) are new components of the miRNA degradation complex. Using the power of the nematode C. elegans and molecular biology, we characterized the role of the loss of function of PPM-2 and HECD-1 in small non-coding RNA pathways. Loss of this phosphatase induces developmental defects which are associated with a defect in the miRNA pathway. Genetically, the phosphatase mutant exacerbates the phenotypes that are observed in animals where the miRNA pathway is affected. Interestingly, we further observed that the loss of the phosphatase affects other small non-coding RNA pathways like the piRNA and the siRNA pathways. At the molecular level, we observed a decrease in the expression level of many Argonaute proteins in phosphatase mutant animals. Upon blocking proteasomal degradation with MG132, we noticed that Argonaute proteins are sent to proteasomal degradation in phosphatase mutant animals. Concerning HECD-1, we noticed that the loss of function of the E3 ubiquitin ligase leads to the decrease of progeny and embryonic lethality due to defects in gametogenesis. Moreover, we observed an accumulation of functional miRNAs. This protein can be implicated in transcription or turnover of miRNAs. VIIn conclusion, our data suggest that PPM-2 controls the stability of Argonaute proteins by sending them through an alternative degradation pathway and that HECD-1 could be implicated in miRNA regulation by modulating their transcription or degradation. My doctoral work helped to highlight a new modulator of small non-coding RNAs, PPM-2, which acts through the regulation of Argonaute protein. A better understanding of the mechanisms controlling the stability and the function of these strong regulators will be useful to develop new therapies.