Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed Sept. 21, 2009). Additional advisors: Martha W. Bidez, W. Michael Carson, Richard A. Gray, W. William Wilson. Includes bibliographical references.

Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.

Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5 can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...

Différentes translocations génomiques sont fréquemment associées à l'apparition de leucémies myéloïdes aiguës (LMA). Ces translocations génomiques résultent de l’assemblage de deux gènes conduisant à la production d'une protéine de fusion. C'est le cas de la translocation t (3; 5) (q25.1; q34) impliquant le suppresseur tumoral NPM et l'oncogène MLF1 donnant naissance à la protéine de fusion NPM-MLF1. Généralement, les gènes impliqués dans ces translocations contrôlent la croissance cellulaire, la différenciation ou la survie cellulaire. Cependant, pour NPM-MLF1 les causes du gain ou de la perte de fonction associée à la translocation demeurent inconnues car nous ne savons pas comment cette translocation peut favoriser ou participer à l'avènement de la LMA. Le but de ce travail est d’analyser le rôle de NPM-MLF1 dans le cancer et d’examiner comment son activité contribue à la leucémie en faisant des études d’interactions protéine/protéine. En effet, l’étude de la fonction d’une protéine implique souvent de connaître ses partenaires d’interactions. Pour ce faire, la technique de double hybride dans la souche de levure AH109 a été utilisée. Tout d’abord, les ADN complémentaires (ADNc) de MLF1, NPM1 et de NPM-MLF1, MLF1-Like (une partie de MLF1 de l’acide aminé 94 à 157) normaux et mutés du domaine MTG8-Like constitué des acides aminés (a.a.) 151 à 164 de MLF1 (excepté NPM) ont été clonés dans un vecteur d'expression de levure pGBKT7. Les ADNc de GFI-1, mSin3A, PLZF, HDAC1 et HDAC3 ont été clonés dans le plasmide pGADT7 de façon à créer des protéines de fusion synthétiques avec le domaine de liaison à l'ADN et de trans-activation de la protéine GAL4. Le plasmide pGBKT7 possède un gène TRP1 et pGADT7 un gène LEU2 qui permettent la sélection des clones insérés dans la levure. Aussi, le pGBKT7 a un épitope c-myc et pGADT7 un épitope HA qui permet de voir l’expression des protéines par buvardage de type Western. Après la transformation des levures les interactions protéine/protéine ont été observées en vérifiant l’expression des gènes rapporteurs HIS3, LacZ, MEL1, ADE2 de la levure en utilisant des milieux de sélection YPD/-Leu/-Trp, YPD/-Leu/-Trp/-His, YPD/-Leu/-Trp/-His/-Ade, YPD/-Leu/-Trp/+ X-Gal, YPD/-Leu/-Trp/ + X-α-Gal. Ensuite, les interactions trouvées par double-hybride ont été vérifiées dans les cellules érythroleucémiques K562 par immuno-précipitation (IP) de protéines suivies de buvardages Westerns avec les anticorps appropriés. NPM-MLF1, MLF1, MTG8, MLF1-Like surexprimés dans les cellules K562 ont été clonés dans le plasmide pOZ-FH-N. pOZ-FH-N possède un récepteur IL-2 qui permet de sélectionner les cellules qui l’expriment ainsi qu’un tag Flag-HA qui permet de voir l’expression des protéines par buvardage-Western. Les résultats du double-hybride suggèrent une interaction faible de NPM-MLF1 avec HDAC1, HDAC3 et mSin3A ainsi qu’une interaction qui semble plus évidente entre NPM-MLF1 et PLZF, GFI-1. NPM interagit avec GFI-1 et mSin3A. Aussi, MLF1 et MLF1-Like interagissent avec HDAC1, HDAC3, GFI-1, PLZF mais pas avec mSin3A. Les IP suggèrent que NPM-MLF1 interagit avec HDAC1, HDAC3, mSin3A et PLZF. MLF1 et MLF1-Like interagissent avec HDAC1, HDAC3 et mSin3A. L’interaction de NPM-MLF1 avec GFI-1, MLF1 et MLF1-Like avec PLZF et GFI-1 n’a pas encore été vérifiée par IP. Ainsi, nos observations permettent de suggérer que NPM-MF1, MLF1 et NPM pourraient jouer un rôle dans la transcription et la régulation de l’expression de certains gènes importants dans l’hématopoïèse et une variété de processus cellulaires parce qu’ils interagissent avec différents corépresseurs. En déterminant les partenaires protéiques de MLF1, NPM et NPM-MLF1, leurs fonctions et comment NPM-MLF1 influence et modifie le fonctionnement cellulaire normal; il sera possible de renverser le processus de LMA favorisé par la t (3; 5) NPM-MLF1 par la technologie d’interférence à l’ARN. / Abstract Different genomic translocations are frequently associated with the development of acute myeloid leukemia (AML). Genomic translocations can result in the fusion of two genes leading to the formation of a fusion protein. This is the case of the T (3; 5) (q25.1; q34) translocation that implicates the tumour suppressor NPM1 and the oncogene MLF1, giving rise to the fusion protein NPM-MLF1. Generally the genes implicated in these translocations control cell growth, differentiation and survival. However, for NPM-MLF1 the reasons behind the gain or loss of function associated with the translocation are still unknown as we still ignore how this translocation can enhance or take part in the AML development. The goal of my master degree project was to analyse in part the role of NPM-MLF1 in cancer and to examine how its activity can contribute in leukemia through protein/protein interaction assays. The usual study of a protein function implicates the investigation of interacting partners. For this purpose, we used the yeast AH109 to conduct a two-hybrid screen assay. The MLF1, MLF1-Like (amino acid 94 to 157 of MLF1), NPM1 and NPM-MLF1 cDNAs, normal and mutated in the MTG8-Like domain from the amino acid (a.a.) 151 to 164 of MLF1 (with the exception of NPM1) were cloned into the yeast expression vector pGBKT7. The GFI-1, mSin3A, PLZF, HDAC1 and HDA3 cDNAs were cloned into the vector pGADT7. These clones were developed to creat synthetic fusion proteins with the DNA binding or trans-activation domain(s) of the protein Gal4. The pGBKT7 vector contains the TRP1 gene and the pGADT7 the LEU2 gene. These genes were used for the selection of the yeast clones transformed with the plasmids mentioned above. In addition, the pGBKT7 vector has c-myc-tag and the PGADT7 vector the HA-tag allowing the assessment of protein expression through Western Blot analysis. After yeast transformation, the protein/protein interaction were studied while verifying the expression of the reporter genes HIS, LacZ, MEL1, ADE while using the following selective medias YPD/-Leu/-Trp, YPD/-Leu/-Trp/-His, YPD/-Leu/-Trp/-His/-Ade, YPD/-Leu/-Trp/+ X-Gal, YPD/-Leu/-Trp/ + X-α-Gal. The interactions determined by the two-hybrid screening were verified in the erythroleukemic cells K562 using immuno-precipitation (IP) of the proteins followed by western blot using the appropriate antibodies. To achieve this, NPM-MLF1, MLF1, ETO, MLF1-Like cDNAs were cloned into the pOZ-FH-N vector that possess an IL2 receptor, which allows the selection of the positive transformed clones in the cell and a Flag-HA tag that permit the verification of protein expression through Western-blot. The two-hybrid screen results suggest that NPM-MLF1 interacts with HDAC1, HDAC3, PLZF, GFI and mSin3A. NPM interacts with GFI-1 and mSin3A. This has not been yet verified using the IP method. As in the case of MLF1, MLF1-Like interacts with HDAC1, HDAC3, GFI-1 and PLZF. However, no interaction was observed with Sin3A. The IP experiments suggest that NPM-MLF1 interacts with HDAC1, HDAC3, mSin3A and PLZF. MLF1 and MLF1-Like interact with HDAC1, HDAC3 and mSin3A. The interaction of NPM-MLF1 with GFI1 as well as MLF1 and MLF1-Like with PLZF and GFI-1 are not yet verified by IP. Therefore, our observations led to the suggestion that NPM-MLF1, MLF1 and NPM can play a role in the transcription and the regulation of the expression of certain genes that are important for hematopoiesis and a variety through the determination of the protein partners of MLF1, NPM and NPM-MLF1, their functions and how NPM-MLF1 influence/modify the normal cellular function, and by focusing on this study, it might become possible to reverse the AML process that is by the t(3;5) NPM-MLF1 while using the RNA interference technology.

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