Abstract not available

Thesis (M.Tech.-Medical Technology)--Cape Technikon, 1997. / Bibliography: leaf 96-114. Also available online.

Résumé Le complexe V mitochondrial (F1FO-ATP synthétase) catalyse la phosphorylation de lADP par le phosphate inorganique en utilisant la force proton-motrice générée par la chaîne de transport délectrons. Ce complexe protéique possède deux domaines : un secteur associé à la membrane, FO, impliqué dans la translocation des protons, et un domaine extrinsèque, F1, qui catalyse la synthèse dATP. Les deux secteurs sont connectés par deux bras : un bras central qui couple la translocation des protons à la région catalytique, et un bras latéral qui est considéré comme faisant partie du stabilisateur (stator) de lenzyme. Au cours de ce travail, nous nous sommes intéressés à lenzyme de deux algues appartenant à la classe des Chlorophyceae, Chlamydomonas reinhardtii et Polytomella sp.. L'enzyme des deux algues présente une composition sous-unitaire atypique, les sous-unités classiquement retrouvées chez les eucaryotes et impliquées dans larchitecture du bras périphérique ou dans la dimérisation du complexe en étant absentes. En contrepartie, 9 sous-unités dorigine évolutive inconnue sont associées à lenzyme. Elles ont été appelées Asa1 à 9 pour ATP Synthase Associated protein. Chez C. reinhardtii et Polytomella sp., lATP synthétase présente une stabilité accrue de sa forme dimérique in vitro, et, in vivo, les cellules de C. reinhardtii sont insensibles à loligomycine, un puissant inhibiteur de la translocation de protons au travers de FO. Nous avons dans un premier temps tenté détablir la composition sous-unitaire du complexe V chez des espèces appartenant aux différentes classes de Chlorophytes (Chlorophyceae, Trebouxiophyceae, Prasinophyceae et Ulvophyceae) en combinant analyses génomiques et protéomiques. Plusieurs sous-unités Asa ont ainsi pu être détectées chez des algues appartenant à divers ordres de Chlorophyceae. Au contraire, les analyses de séquences disponibles chez les autres classes de Chlorophytes (Trebouxiophyceae, Prasinophyceae et Ulvophyceae) indiquent une composition canonique de lenzyme. Lanalyse de la stabilité de la forme dimérique du complexe de différentes espèces d'algues vertes sur BN PAGE (Blue Native PolyAcrylamide Gel Electrophoresis) suggère également que la présence dun dimère stable est caractéristique aux Chlorophyceae. Par ailleurs, leur croissance, respiration, et niveaux d'ATP sont à peine affectés par la présence d'oligomycine à des concentrations inhibitrices chez les représentants des autres classes de Chlorophytes. Les nombreuses particularités communes aux algues appartenant à cette classe suggèrent que la perte d'éléments canoniques du stator est apparue lors de la séparation des Chlorophyceae et a été accompagnée du recrutement de nouvelles sous-unités. Ce réarrangement drastique de la composition de stator et du module de dimérisation pourrait avoir conféré de nouvelles propriétés à lenzyme, notamment une meilleure stabilité et une plus grande résistance à loligomycine. Nous avons également étudié la fonction de la sous-unité atypique Asa7 en inactivant son expression chez C. reinhardtii. Bien que la perte de la sous-unité Asa7 n'aie aucun impact sur la bioénergétique des cellules ou sur la structure mitochondriale, elle déstabilise lenzyme in vitro et rend la croissance, la respiration, et de le niveau d'ATP sensible à oligomycine. L'impact de la perte de l'activité ATP synthétase mitochondriale chez un organisme photosynthétique a été étudié chez C. reinhardtii par linactivation de l'expression du gène ATP2, codant pour la sous-unité catalytique beta. Les résultats démontrent que, en l'absence de beta, l'ATP synthétase ne peut plus être assemblée et les cellules deviennent dépendantes de la photosynthèse. La respiration en présence ou en absence du découpleur CCCP suggère que le passage des protons à travers la membrane interne mitochondriale est bloqué chez la souche mutante. Enfin, la morphologie des mitochondries est affectée, et les chloroplastes montrent un réaménagement massif de l'appareil photosynthétique, suggérant des répercussions importantes sur la synthèse dATP par les chloroplastes. Ces résultats contribuent à la compréhension des interactions entre organites bioénergétiques chez les organismes photosynthétiques.

Pantothenate synthetase (PS), which catalyzes the last step in the pantothenate (vitamin B5) biosynthesis, is a dimeric enzyme present in bacteria, fungi and plants. The enzymatic properties of PS from Escherichia Coli, Mycobacterium tuberculosi, Fusarium Oxysporum, Lotus japonicus, Oryza sativum, Brassica napus and Arabidopsis thaliana have been characterized. The chemical reaction and the proposed mechanism of reaction are identical for PS, irrespective of the source. However, from an enzyme mechanistic point of view, plant PS’s are dissimilar to their bacterial counterparts, in that they exhibit “allosteric behavior”, a property that has not been observed in the bacterial enzyme. The behavior is quite remarkable when one takes into consideration the fact that plant PS’s share a high degree of sequence identity (~ 40%) with the bacterial enzymes. Even more intriguing is the structural mechanism proposed to explain the observed differences in structure between the PS’s from E.Coli and M.tb, which share a 42% sequence identity. Till date there is no structural information available on the plant PS’s and of the substrate bound conformation of E.coli PS. This thesis aims to provide an understanding on some aspects of the structure – function relationship of this physiologically important enzyme. Specifically, the solution properties of E. coli PS have been examined using high-resolution multinuclear, multidimensional NMR methods. Given the large size of the full-length protein (~ 63 KDa), the structurally distinct N and C-terminal domains were cloned and expressed as individual proteins and their properties investigated. Towards this end, the tertiary fold of the 40 kDa dimeric amino-terminal domain of E. coli pantothenate synthetase has been determined using multidimensional multinuclear nuclear magnetic resonance (NMR) methods (PDB entry 2k6c). Sequence specific resonance assignments for backbone HN, 15N, 13Cα, 13C', sidechain 13Cβ and aliphatic 13CH3 (of isoleucine, leucine and valine residues) were obtained using perdeuterated ILV-methyl protonated samples (BMRB entry 6940). Secondary structure of nPS was determined from 13C secondary chemical shifts and from short and medium range NOEs. Global fold of the 40 kDa homo-dimeric nPS has been determined using a total of 1012 NOEs, 696 dihedral angles, 260 RDCs, 155 hydrogen bonds, radius of gyration potential, non-crystallographic symmetry potential and database derived potential based upon the Ramachandran map. The calculated structures, which show that the N-terminal domain forms a homo-dimer in solution, is of high stereochemical quality as judged by the Ramachandran statistics (70% of the residues have backbone dihedral angles in the allowed region, 25.5% in the additionally allowed region, 4.0% in generously allowed region, and only 0.5% in disallowed region). Dynamics of nPS, which has rotational correlation time τc of 17.3 ns, was investigated by 15N relaxometry measurements. Results of these studies indicate that the E. coli protein exhibits dynamic nature at the dimer interface. These structural and dynamic features of the protein were found to be of interest when correlated with NMR based substrate binding studies. Interaction of homo-dimeric amino-terminal domain (nPS) of E. coli pantothenate synthetase (PS; encoded by the gene panC; E.C. 6.3.2.1) with the substrates pantoate, β-alanine, ATP and the product pantothenate has been studied using isotopically edited solution NMR methods. Addition of pantoate prior to ATP has led to the interesting observation that pantoate binds each monomer of nPS at two sites. ATP displaces a molecule of pantoate from the ATP binding site. β-alanine and pantothenate do not bind the protein under the condition studied. Binding of pantoate and ATP also manifests as changes in residual dipolar couplings (RDCs) of backbone 1H-15N pairs in nPS when compared to the free form of the protein. Structures of homo-dimeric nPS bound to two molecules of pantoate (PDB entry 2k6e) as well as to pantoate + ATP (PDB entry 2k6f) were calculated by inclusion of hydrogen bonds between the ligands and backbone 1H-15N pairs of nPS in addition to other NMR derived restraints. The ligand bound structures have been compared to the similar forms of the M. tb PS. Structure of each monomer of nPS bound to pantoate and ATP shows the substrates in a favorable orientation for the intermediate pantoyl adenylate to form. Moreover, at all stages of substrate binding the symmetry of the dimer was preserved. A single set of resonances was observed for all protein-ligand complexes implying symmetric binding with full-occupancy of the ligands bound to the protein. In an effort to understand the structural basis of the observed enzymatic properties of plant PS’s, a structural model of the Arabidopsis PS was constructed. The results of these structural and substrate binding studies strongly suggest that 1 Substrate binding to PS occurs only at the active site. 2 There are no additional substrate binding sites which could potentially participate as regulatory sites. 3 Pantoate does not bind at the dimer interface to induce the observed homotropic effects. 4 The structural results presented on the substrate bound forms of nPS have direct implication for the development of novel antibacterial and herbicidal agents. Recently a great deal of interest has been evinced on the effects of molecular crowding on protein folding / unfolding pathways. Nuclear magnetic resonance is the only method by which high resolution structural information can be obtained on partially denatured states of a protein under equilibrium condition. Recent methodological advances have enabled the determination of high resolution structures using information embedded in the residual dipolar couplings. Molecular crowding using confinement may thus reveal important details about chaperone mediated protein folding. We have attempted to develop a protocol to study the effects of molecular confinement by sequestering proteins in poly-acrylamide gels and then subjecting these protein molecules to denaturation and then characterize these states by nuclear magnetic resonance. The preliminary results of these studies are described here.

The bacterial cell needs ammonia for synthesis of glutamine from glutamate. Only one enzyme is able to catalyze this reaction, namely glutamine synthetase (GS). GS can be regulated both transcriptionally and post-translationally and it is present in all kingdoms of life. Our study has been focused on the post-translational regulation of GS in the diazotrophic bacterium Rhodospirillum rubrum. A number of proteins are involved in the covalent regulation of GS, among them are the regulatory PII proteins that depending on growth conditions also like GS are covalently modified. We have purified all proteins involved in GS regulation and developed several in vitro assays with the aim of understanding GS regulation in R. rubrum. Studies on the influence of the small metabolite effectors α-ketoglutarate and glutamine are also included together with the effect of divalent cations. In both R. rubrum and Escherichia coli, one of the enzymes participating in GS regulation is the bifunctional enzyme GlnE. GlnE is responsible for both the attachment and the removal of AMP groups from GS, which basically leads to a more inactive or active enzyme respectively. Apart from examining the above functions of GlnE, we have also found a novel third activity of R. rubrum GlnE, an antioxidant function, which is located in the C-terminal domain. We have examined this novel activity of GlnE in great detail, including site specific mutagenesis. We also generated and analyzed ΔglnE mutants in R. rubrum and the results from these studies show that suppressor mutations can occur within glnA, the gene encoding GS. We assume that the function of these suppressor mutations is to lower the specific activity of GS, which otherwise might be too high in a ΔglnE mutant since they lack the ability to adenylylate GS. In other words, it seems that ΔglnE mutants can not be generated without producing suppressor mutations.

The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.

As shown by recent reports, number of obese people in recent years has been on the increase, there are about 4 million people in Taiwan who are considered to be overweight. World Health Organization (WHO) and United States Center for Disease Control and Prevention (CDC) publicly announced that: Obesity will be the greatest health killer of this century, its damage to personal health is comparable to that of cigarettes. Obesity can cause heart problems, diabetes, artery diseases, high blood pressure, increased chances of cancer occurrence, condition increase and deteriora- tion of Alzheimer¡¦s disease, gall bladder diseases, and shortening of life span. The cause of obesity is due to a fault in adipocytes metabolism functions, and because of this, research into adipocytes molecular regulation is becoming more popular and valued. The process of adipogenesis, the formation of adipose tissue, has become better understood by the studies of several cell types that can be induced to undergo differentiation into adipocytes. The first, and the best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer -binding protein £] (C/EBP£]). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBP£_. Immediately after exposure to exogenous inducers, the gene expression of C/EBP£] and C/EBP£_ significantly and transiently increases, C/EBP£] and C/EBP£_ may also regulate the expression of C/EBP£\ and PPAR£^. C/EBP£\ and PPAR£^ are considered to play a prominent role in regulating the gene expression of proteins necessary for the development fo the functional mature adipocyte. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBP£\ and PPAR£^ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin. The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. My research is aimed at investigating the adipocytes differentiation and regeneration adaptive mechanisms of mice 3T3L-1 preadipocytes and human processed lipoaspirate cells (PLA). By using adipocytes culture techniques in conjunction with adipocytes growth induction and gene delivery techniques to further study obesity related genes, POMC and PTEN, and downstream regulators , PPAR£^ and Adiponectin, in regards to their roles in the process of adipocytes differentiation.

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The protein Liquid facets (Lqf) promotes endocytosis at the plasma membrane1. Lqf activity is required for proper Notch signaling, likely through facilitating the endocytosis of Notch ligand by indirectly linking ligand to clathrin. A genetic modifier screen to identify genes that interact with lqf was performed by a previous graduate student. Genes identified in the screen might provide new insights into how Lqf promotes endocytosis or how Notch signaling is regulated. In this work, I performed genetic mapping techniques to identify the genes mutated in each complementation group of the screen. I identified the gene mutated in complementation group 6 as mitochondrial alanyl tRNA synthetase (Aats-ala-m). tRNA synthetases link a tRNA to its cognate amino acid during translation. Mitochondrial tRNA synthetases function in the mitochondria in translation. Aats-ala-m genetically interacts with lqf, suggesting the two genes function in the same pathway. In this work, I also identified chromosomal regions where the genes mutated in complementation groups 1,2, and 9 are located. / text

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