Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The phosphatidylinositol polyphosphates play a fundamental role in intracellular signalling. Of particular importance is phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃], which acts by recruiting effector proteins to the cell membrane. PtdIns(3,4,5)P₃ interacts with its protein targets through selective binding domains that include the pleckstrin homology (PH) domain. The PH-domain-containing kinase, protein kinase B (PKB/Akt), which interacts with PtdIns(3,4,5)P₃, is upregulated in ~15 human malignancies. Significantly, inhibition of the PtdIns(3,4,5)P₃-PKB interaction has proved viable as a point of therapeutic intervention. There is currently a lack of small molecule probes that selectively interact with a given PH domain. Consequently, it is impossible to dissect the cellular function of PH-domain-containing proteins at a molecular level. To address this problem, we have designed and synthesised a number of derivatives of the PtdIns(3,4,5)P₃ inositol head-group – Ins(1,3,4,5)P₄. Replacement of the 5-position phosphate with a range of phosphate bioisosteres afforded compounds that displayed no binding affinity for the PH-domain of general receptor for phosphoinositides 1 (GRP1). However, it was shown that the 5-position sulfamate analogue displayed selectivity for the PH-domain of PKB. The methylphosphate biosiostere at the 1-position displayed binding for both the GRP1 PH-domain as well as the PKB PH-domain. These results demonstrate that subtle modification of the Ins(1,3,4,5)P₄ structure allows the synthesis of compounds that interact selectively with a given PH domain. We will now use these results for the synthesis of a second generation of compounds with improved PH-domain affinity and selectivity.

Phospholipase Cβ (PLCβ) cleaves phosphatidylinositol-4,5-bisphosphate (PIP₂) into the second messengers inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ increases intracellular Ca²⁺, while DAG remains in the membrane, and together with increased Ca²⁺, activates protein kinase C (PKC). PLCβ has low basal activity but is activated following stimulation of G_i- and G_q-coupled receptors through direct interactions with Gα_q and Gβγ. PLCβ is essential for normal cardiomyocyte and vascular smooth muscle function and regulates cell proliferation, survival, migration, and differentiation. However, increased PLCβ activity and expression results in arrhythmias, hypertrophy, and heart failure. PLCβ must interact with the cell membrane for its activity. While heterotrimeric G proteins stimulate PLCβ, they are insufficient for full activation, suggesting the membrane itself contributes to increased lipid hydrolysis, potentially via interfacial activation. However, how the composition of the membrane and its resulting properties, such as surface charge, contribute to adsorption and interfacial activation is not well-established. Furthermore, whether or how interfacial activation also impacts other regulatory elements in PLCβ and Gα_q-dependent activation is unknown. Using an innovative combination of atomic force microscopy on compressed lipid monolayers and biochemical assays, we are beginning to understand how the membrane itself, PLCβ autoinhibitory elements and Gα_q regulate PLCβ activation. These studies provide the first structure-based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.

Actively proliferating cells constantly monitor and re-adjust their metabolic pathways to ensure the replenishment of phospholipids necessary for membrane biogenesis and intracellular trafficking. In Saccharomyces cerevisiae, multiple studies have suggested that lysine acetylation has a role in coordinating phospholipid metabolism, yet its contribution towards phospholipid homeostasis remains uncharacterized. In this study we undertook a genetic screen to explore the connection between lysine acetylation and phospholipid homeostasis. We found that mutants of the lysine acetyltransferase complex, NuA4, shared a negative genetic interaction with a mutant of Sec14, a lipid-binding protein that regulates Golgi phospholipid composition. Through transcriptome, genetic, cell biology, and chemical analysis, we discovered that the growth defects between NuA4 and Sec14 mutants is likely derived from impaired fatty acid biosynthesis suggesting a role for NuA4 as a positive regulator of fatty acid biosynthesis. Secondly, we discovered that acetylation on the conserved lysine residue K109 inhibits the localization and function of the Oxysterol-Binding Protein Osh4- a lipid-binding protein that antagonizes the function of Sec14 at the Golgi. Furthermore, regulation of Oxysterol-Binding Proteins by acetylation may be a conserved mechanism as we found that Osh1, a homologue of Osh4, was also acetylated on the equivalent lysine residue. Altogether, we have demonstrated that lysine acetylation can target multiple different phospholipid metabolic pathways which implies that it has a very important role for the regulation of phospholipid homeostasis.

Chez les plantes, l'acide salicylique (SA) a un rôle central dans la réponse à de nombreuses contraintes environnementales et lors du développement. Cependant les événements de signalisation précoces qu'il déclenche sont peu connus. Nous montrons, par marquage métabolique au 33Pi sur une suspension cellulaire d'Arabidopsis thaliana, que le SA induit une diminution rapide et précoce d'un pool de phosphatidylinositol (PI). Celle-ci est accompagnée d'une accumulation de PI 4-phosphate et PI 4,5-bisphosphate. Ces changements sont inhibés par de la wortmannine à 30 μM mais pas à 1 μM, ce qui implique une activation de PI 4-kinase de type III. C'est pourquoi une étude des effets de la wortmannine sur les modifications de transcriptome par le SA a été menée à l'aide de la puce " Complete Arabidopsis Transcriptome MicroArray " (CATMA). Sur 773 gènes régulés par le SA, 112 sont sensibles à 30 μM de wortmannine. En parallèle, nous voyons que l'acide phosphatidique issu de la phospholipase D (PLD) est important pour la réponse génique précoce au SA. Une expérience de puces menée pour identifier les gènes régulés par la PLD en réponse au SA a révélé que parmi 1327 gènes régulés par le SA, 97 gènes sont régulés positivement, et 117 gènes négativement, par la PLD. Les régulons de la voie sensible à la wortmannine et de la voie PLD se chevauchent fortement, ce qui suggère que les deux activités agissent en synergie dans la même voie de signalisation en réponse au SA.

Regulated secretion of hormones, digestive enzymes and other biologically active molecules requires formation of secretory granules. However, the molecular machinery required for secretory granule biogenesis is incompletely understood. I used powerful genetic approaches available in the fruit fly Drosophila melanogaster to investigate the factors required for biogenesis of mucin-containing ‘glue granules,’ which form within epithelial cells of the third-instar larval salivary gland. I discovered that clathrin and the clathrin adaptor protein complex (AP-1), as well the enzyme type II phosphatidylinositol 4-kinase (PI4KII), are indispensable for glue granule biogenesis. Clathrin and AP-1 are necessary for maturation of exocrine, endocrine and neuroendocrine secretory granules in mammalian cells. I found that Drosophila clathrin and AP-1 colocalize at the TGN and that clathrin recruitment requires AP-1. I further showed that clathrin and AP-1 colocalize with secretory cargo at the TGN and on glue granules. Finally, I demonstrated that loss of clathrin or AP-1 leads to a profound block in secretory granule biogenesis. These findings establish a novel role for AP-1/clathrin-dependent trafficking in the formation of mucin-containing secretory granules. Type II phosphatidylinositol 4-kinase (PI4KII) generates the membrane lipid phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network and is required to recruit cargo to endosomes in mammalian cells. I generated null mutations in the sole Drosophila PI4KII and demonstrated a role for PI4KII in both glue granule and pigment granule biogenesis. PI4KII mutant salivary gland cells exhibit small glue granules and mislocalize glue protein to abnormally large late endosomes. Additionally, PI4KII mutants exhibit altered distribution of the granule specific SNARE, SNAP-24. These data point to a crucial role for PI4KII in sorting of regulated secretory products during granule biogenesis. Together, my results indicate that the larval salivary gland is a valuable system for investigating molecular mechanisms involved in secretory granule biogenesis, and provide a framework for future studies using this system.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 132-151). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 105-135).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 124-147).

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