The NADP-dependent trifunctional methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase-Synthetase (DCS) is responsible for the interconversion of one-carbon substituted tetrahydrofolates (THFs) required for methylation reactions and nucleotide synthesis in the cytoplasm of mammalian cells. A spontaneously immortalized DCS null fibroblast cell line was found to be a purine auxotroph due to a lack of 10-formylTHF required for de novo purine synthesis (Christensen et al. 2005). Using a retroviral infection system the DCS null fibroblasts were infected with constructs designed to express wild type DCS or proteins with D and S activities inactivated by point mutations. Western analysis and activity assays confirmed protein expression. All constructs rescued the cell line from purine auxotrophy showing that one-carbon substituted THF derived from cytoplasmic serine and mitochondrial formate can be utilized in purine synthesis. Supported by previous studies, radiolabeling experiments tracing incorporation of exogenous 3-14C serine and 14C formate demonstrated that mitochondrial formate is the preferred source of one-carbon units for purine synthesis in these cells.

This thesis examines the molecular properties of Nramp proteins by centering on the two mammalian orthologs. Nramp1 (Slc11a1) is expressed in phagocytic cells and restricts replication of intracellular pathogens by removing divalent metals from the phagolysosome. Nramp2 (DMT1, Slc11a2) mediates uptake of dietary iron in the duodenum and aids in the acquisition of transferrin-associated iron in many cell types. The first half of this thesis explores structure-function relationships. In Chapter 2, the role of charged amino acids within the membrane-spanning segments of Nramp2 was examined by site-specific mutagenesis. These studies identified several invariant charged residues essential for metal transport and pH regulation of activity. In Chapters 3 and 4, the effects of two NRAMP2 mutations found in human patients suffering from severe congenital hypochromic microcytic anemia and iron overload were characterized in vitro. The first mutation was an E399D substitution in a region known as the "conserved transport motif" of the protein. The second mutation was an R416C substitution at an invariant residue in TM9. The effects of both mutations on expression, activity, and subcellular targeting were characterized. In both cases, a quantitative reduction in Nramp2 expression was found to be the cause of microcytic anemia and iron overload in the patients. The second half of this thesis focuses on the subcellular targeting of Nramp1 and 2. In Chapter 5, cytoplasmic signal(s) in Nramp2 responsible for its subcellular targeting/internalization from the plasma membrane were studied. This work led to the identification of a tyrosine-based motif in the carboxyl terminus of Nramp2 (YLLNT555-559) critical for the transporter's internalization from the cell surface and its recycling back to the plasma membrane. Chapter 6 explored differences in trafficking between two splicing isoforms of Nramp2 and found that one isoform (isoform 1) possessed differences in internalization/recycling which enabeled it to become enriched at the plasma membrane. In Chapter 7, the subcellular trafficking properties of Nramp1, including cytoplasmic sequences responsible for targeting to lysosomes, were investigated by using chimeric Nramp1/Nramp2 proteins. This work led to the identification of a tyrosine-based motif (YGSI15-18) in the amino terminus of Nramp1 that functions as a lysosomal targeting signal.

An empirical rule which predicts which enantiomer will be preferred by lipases (triacylglycerol hydrolases) based on the relative sizes of the substituents has previously been developed for secondary alcohols. This rule was then extended towards primary alcohols for lipase from Pseudomonas cepacia (PCL). However, the rule for primary alcohols shows an opposite enantiopreference compared to the rule for secondary alcohols. Furthermore, it is no longer valid when an oxygen lies at the stereocentre. This thesis explores the molecular basis for the empirical rule for primary alcohols and why the rule is no longer valid when an oxygen lies next to the stereocentre. Molecular modeling studies on 2-methyl-3-phenyl-1-propanol and 2-phenoxy-1-propanol, both as the free substrate and covalently linked to the active site of PCL were performed. The modeling studies were then supported by two series of laboratory experiments: determination of the kinetic constants, kcat and Km; and chemical modification of PCL by acetylation and nitration.

The aim of this project was to study the binding of a series of truncated forms of the propeptide to rat cathepsin B. This was done previously by using enzyme inhibition assays (Chen et al, 1996), in which binding is detectable only for those peptides that bind to the catalytic site, and hence compete with the substrate for that site. For this reason, Isothermal Titration Calorimetry (ITC) was utilised since heat detection provides a more direct measurement of binding, irrespective of its location on the enzyme. However, several difficulties were encountered which rendered this approach less feasible. Nevertheless, in the course of analysing complications of the cathepsin B system, other biophysical techniques were used in addition to ITC. In turn, this helped further characterise several aspects of cathepsin B interactions. / In summary, ITC was used to study cathepsin B interactions with its propeptide, several nitrile inhibitors, leupeptin, and E64. (Abstract shortened by UMI.)

My Master's thesis consists of the structural analysis of two proteins: The first is the structural analysis of Interleukin-1 receptor-associated kinase-4 (IRAK-4). IRAK-4 is a serine/threonine kinase which constitutes a crucial component of the innate immune system. Among the IRAK family members, IRAK-4 is the only one which uses its kinase activity in downstream signal transduction pathways. IRAK-4 also plays a role in mediating adaptive immunity. An IRAK-4 deficiency in humans can cause abnormal inflammatory responses, autoimmune disorders and cancer, making it a potential drug target. A structure of the active form of the isolated IRAK-4 kinase domain has already been published. The goal of my project was to express in insect cells, purify and crystallize the inactive form of full-length IRAK-4 as well as its isolated kinase domain, in order to obtain structural information for this important kinase. Crystals of the non-phosphorylated and inactive form of the IRAK-4 kinase domain were obtained under many conditions. Preliminary diffraction analysis indicated that the crystals are made of protein but diffract X-rays very poorly. The second part of my project involves the structural analysis of the S209E mutant form of murine eIF4E, which can potentially mimic phosphorylation at this site. eIF4E is a eukaryotic initiation factor that binds the mRNA 5'-cap and is an essential component of the 40S ribosomal protein synthesis initiation complex. eIF4E can be phosphorylated by the Ser/Thr kinase Mnk1 in vivo at serine 209, which is thought to affect its ability to bind the cap structure. However, the precise function of this phosphorylation is not clear as different research groups have produced conflicting results. In this study, we focus on the structural analysis of the S209E mutant because it is difficult to obtain phosphorylated eIF4E in vitro. Crystals of the S209E eIF4E mutant formed under several conditions, one of which produced crystals of large enough d / Mon mémoire de maîtrise est composé de l'analyse structurelle de deux protéines : La première partie est l'analyse de la structure de « Interleukin-1 receptor-associated kinase-4 (IRAK-4) ». IRAK-4 est une sérine/thréonine kinase qui constitue une partie essentielle du système immunitaire inné. Parmi les membres de la famille des IRAKs, IRAK-4 est le seul qui utilise son activité kinase dans la cascade de signalisation en aval. IRAK-4 joue aussi un rôle important dans l'immunité adaptive. Une déficience en IRAK-4 chez les humains peut causer des réponses inflammatoires abnormales, des désordres auto-immunitaires ainsi que le cancer, ce qui fait de IRAK-4 une potentielle cible thérapeutique. La structure de la forme active du domaine kinase d'IRAK-4 a déjà été publiée. Le but de mon projet était d'exprimer dans des cellules d'insecte la forme inactive d'IRAK-4 en sa longueur complète, ainsi que le domaine kinase isolé, afin de les purifier et de les cristalliser pour obtenir de l'information structurelle sur cette kinase essentielle. Des cristaux de la forme inactive et non phosphorylée du domaine kinase d'IRAK-4 ont été obtenus sous différentes conditions. L'analyse préliminaire par diffraction de rayons X indique que les cristaux sont constitués de protéine mais diffractent très pauvrement. La seconde partie de mon projet implique l'analyse structurelle du mutant S209E de murine eIF4E, ce qui a le potentiel d'imiter la phosphorylation à ce site. eIF4E est un facteur eukaryote d'initiation de translation qui se lie au 5'-cap de l'ARN messager et est un constituant essentiel du complexe ribosomal 40S d'initiation de synthèse de protéine. eIF4E peut être phosphorylé par Mnk1 in vivo à la sérine 209, ce que l'on croit pourrait affecter sa capacité de se lier au 5'-cap. Cependant, la fonction précise de cette phosphorylation n'est toujours pas claire car différents groupes de chercheurs ont obtenu des résultats contradic

We have initiated a research program intended to prepare lipid soluble free radical sensitive fluorescent probes which can readily report the antioxidant activity within the lipid bilayers. This work provides a substantial amount of information useful in the rational design of pyrromethane dye-based off-on fluorescent probes/switches for redox visualization. In this thesis we report the photophysical properties of a newly prepared Trolox®-PM605 dye (B-TOH) which exhibits a marked off-on fluorescence behaviour in the presence of radicals in homogeneous and microheterogeneous solutions. The absorption and fluorescence spectra, fluorescence quantum yields, and fluorescence lifetimes for this probe when dissolved in organic solvents, as well as in dimirystoyl phosphatidylcholine (DMPC) lipid vesicle suspensions in water are reported herein. The analogous photophysical properties were measured for PM605, the precursor fluorophore used in B-TOH synthesis, its deacylated derivative PM-OH, and a control probe 3,5-Di-tert-butyl-4-hydroxybenzoic acid-PM605 (PM-BHB, which is expected to have a lower reactivity towards radicals) adduct whose synthesis is further described herein. We also report the emission time profile of B-TOH, PM-BHB, and PM-OH following their prolonged exposure to peroxyl radicals in organic solvents and liposome water suspensions. Via careful electrochemical analysis and HPLC oxidation product studies, the observed emission growth of B-TOH is shown to occur via the deactivation of a Photoinduced Electron Transfer (PET) process, operative only in reduced B-TOH. Following exposure of B-TOH to free radicals and subsequent oxidation, PET is surpressed. In summary, we report a novel hydrophobic fluorescent antioxidant indicator with optimum off/on ratio properties for homogeneous solution studies. Antioxidant depletion and the onset of radical mediated oxidation can be directly monitored following emission enhancement over time, thus dramatically facilitati / Nous avons initié un programme de recherche qui a pour but de développer des sondes fluorescentes, sensibles aux radicaux libres, qui soient solubles dans les solutions lipidiques. Ces sondes devront pouvoir rapporter les activités anti-oxidantes des bi-couches lipidiques. Ce travail fournit des informations importantes pour la conception rationnelle de colorants dérivés du pyrrométhane qui pourront servir d'indicateurs fluorescents "on-off" de la visualisation des réactions redox. Dans cette thèse, nous rapportons les propriétés du nouvel indicateur Trolox-PM605 (B-TOH) qui montre un comportement fluorescent "on-off" marqué en présence de radicaux en solutions homogènes et micro-homogènes. Les spectres d'absorption et de fluorescence, les taux de fluorescence quantique, et la durée de vie de la fluorescence pour cette sonde quand dissoute dans des solvants organiques ainsi que dans des vésicules lipidiques de dimirystoyl de phoshatidylcholine (DMPC) en suspensions dans l'eau sont également rapportées. Les propriétés analogues photophysiques du PM605 (le précurseur fluorophore utilisé pour la synthèse du B-TOH) et du PM-OH (son dérivé acétyle) ainsi que celles d'une sonde contrôle, le 3,5-Di-tert-butyl-4-hydroxybenzoic acid-PM605 (PM-BHB, dont il est attendu d'observer une plus faible réactivité envers les radicaux libres) ont ainsi été mesurées. Nous rapportons également le profil temporel d'émission du B-TOH, du PM-BHB ainsi que du PM-OH, à la suite de leur exposition prolongée aux radicaux peroxydes en solution dans des solvants organiques ou des suspensions aqueuses de liposomes. De précises analyses électrochimiques et des études des produits d'oxidation HPLC ont permis l'observation de la croissance de l'émission du B-TOH, qui se déroule via la désactivation d'un processus de Transfert d'Electron Photo-induit (TEP), ce processus ne s'opérant que dans le contexte du B-TOH réduit. A la suite de l'exposition du B-T

Maltose Binding Protein (MBP) of Escherichia Coli, a kind of periplasmic protein, can bind its ligands interacting predominantly either with their anomeric end (end-on binding) or with the middle of the maltodextrin chain (middle binding). Using NMR spectroscopy, we have studied the modes by which maltose, linear maltodextrin and some derivatives like $ beta$-cyclodextrin bind to MBP. 1D proton difference spectra and 2D HSQC proton-nitrogen correlation spectra were acquired of MBP in the presence of different ligands. Spectra with linear maltodextrins showed many common features and were distinctly different from those of MBP with $ beta$-cyclodextrin. 2D HSQC spectra suggest further that MBP- $ beta$-cyclodextrin adopts an open form conformation similar to that of ligand free MBP because of the surprisingly similarity of their spectra. Ligands such as $ beta$-cyclodextrin, can not be transported into the cyto-plasm but have high affinity for MBP, multiple $ alpha$(1-4) linkages and no reducing end. These ligands bind to MBP mainly by the middle binding mode. This suggests that this mode determines high affinity binding of ligand to MBP, but doesn't produce a physiologically active complex.

Apoptosis is a genetically predetermined cell death program common to multicellular organisms. Bcl-2/BclXL function to suppress apoptosis and promote survival by inhibiting the death pathway at key points. One key point of the death pathway involves the activation of the caspases which are proteolytic enzymes that systematically degrade the cell. Recently, studies have shown that Bap31 is a polytopic integral membrane protein of the ER which forms a complex with Bcl-2/BclXL, a CED-4-like-adaptor-molecule, and initiator procaspase-8. This suggests that the Bap31 complex may regulate apoptosis by bridging core components of the cell death machinery at the ER. / In order to further our understanding of the Bap31 complex in vivo we have isolated it from human KB cells that stably express recombinant forms of Bap31. Bap31 localized to the ER fraction of rat liver cells, and analysis by cryo-immuno-cytochemistry using electron microscope confirmed that Bap31 primarily resides in the ER. Following an adenovirus E1A death signal, the Bap31 complex does not change in size as judged by size exclusion chromatography. Immunoprecipitation of the chromatography isolated complex showed that it consisted primarily of a Bap31 oligomer.

The E4orf4 protein of adenovirus type 5 has been shown to induce p53-independent apoptosis. The only known function of E4orf4 is to bind to the Balpha subunit of protein phosphatase 2A (PP2A). A series of experiments were performed with both wild-type and mutant forms of E4orf4 to examine Balpha subunit binding and cell killing following introduction into p53-deficient cells. Deletion mutant analysis indicated residues 12--18 and 103--114 were not required for cell killing. Point mutant analysis indicated that residues required for Balpha binding and cell killing are dispersed throughout the E4orf4 sequence. Analysis of these results showed that binding to PP2A via its Balpha subunit is essential for induction of p53-independent apoptosis by E4orf4. Binding assays showed that E4orf4 cannot bind B'' subunits of PP2A. In human cells, E4orf4 induces an apoptosic pathway that involves caspase-3 independent cleavage of poly(ADP-ribosyl) polymerase. E4orf4's activity may be controlled through post-translational modification since E4orf4 was found to be phosphorylated on serine residues.

Insulin-like growth factors (IGFs) and stromal cell derived factor 1 (SDF-1) have been suggested as being potential therapeutic agents in HIV infected patients. The cDNA coding for a recombinant fusion protein of IGF 1I and SDF-1 was synthesized by PCR and inserted into a commercial non-lytic insect cell expression system. The successful production of the recombinant protein in the culture medium of insect cells transfected with the recombinant plasmid was monitored using HPLC. Purified protein was characterized by following 3H-thymidine incorporation into bovine fetal liver cells for the identification of IGF II and using Western blots and enzyme-linked immunosorbent assays for the identification of SDF-1. / A modified IGF II-SDF 1 chimera, which included a N-glycosylation site, was also successfully produced by insect cells. Mass spectrometry analysis indicated that the glycosylated and non-glycosylated proteins had molecular masses of 17.6 kDa and 14.5 kDa, respectively. The recovery of the glycosylated protein was higher than that obtained with the non-glycosylated form after purification by heparin affinity chromatography, a property that could be an advantage for the large scale production of the protein. Finally, three additional cDNAs were successfully generated and cloned into insect cell expression vectors. Two of them encoded the glycosylated-and non glycosylated forms of the chimera incorporating a Kozak sequence to improve translation efficiency. The third cDNA encoded a chimera of IGF II and a soluble form of the HIV-binding protein CD4, a potential inhibitor of HIV infection.