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xxi, 98 leaves : ill., tables, graphs ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1978) from the Dept. of Biochemistry, University of Adelaide

xxi, 126 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1978

Thesis (Ph.D. 1978) from the Department of Biochemistry, University of Adelaide.

Thesis (Ph.D.) -- University of Adelaide, Department of Biochemistry, 1978.

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl isomerase enzyme (PPIs) that catalyze the cis-trans inter-conversion of peptidyl-prolyl bonds in proteins. This non-covalent post-translational modification is a reversible mechanism to modulate protein structure and function. PPIs have been implicated in a wide variety of processes from protein folding to signal transduction. Despite these enzymes being ubiquitous, the substrates and functions of most PPIs have yet to be described. FKBP25 is a nuclear FKBP that has been shown to associate with transcription factors and chromatin modifying enzymes, however its functions and substrates remain largely unresolved. FKBP25 is the human ortholog of S. cerevisiae Fpr4, which has been shown to regulate the chromatin landscape by two distinct mechanisms: 1. Acting as a histone chaperone at ribosomal DNA, and 2. Isomerizing histone prolines. Based on these observations, I hypothesized FKBP25 regulates chromatin and/or ribosome biogenesis through isomerization of histone prolines and a discrete collection of substrate proteins. While small molecule inhibitors exist for FKBPs, applying them to dissect the specific function(s) of any given FKBP is confounded by the fact that multiple FKBPs are found in each organism, and several are inhibited by these molecules. In Chapter 2, I biochemically and structurally characterize a set of FKBP25 loss-of-function mutants, yielding a toolset capable of distinguishing between catalytic and non-catalytic functions. These reagents provide the tools necessary to analyze potential substrates of FKBP25 identified in my research going forward. In Chapter 3, I present the first unbiased proteomic screen of FKBP25 associated proteins and show that it interacts with a large number of ribosomal proteins, ribosomal processing factors and a smaller subset of chromatin proteins. I focus on the interaction between FKBP25 and nucleolin, a multi-functional nucleolar protein, and show that FKBP25 interacts with nucleolin and the pre-60s ribosomal subunit in an RNA dependent fashion. In Chapter 4, I gain insight into the role of FKBP25 in ribosome biology, and demonstratex that FKBP25 regulates RNA binding activity of nucleolin, however this does not appear to involve cis-trans prolyl isomerization. Collectively, my work establishes FKBP25 as the first human FKBP to be implicated in the maturation of the pre-60S ribosomal subunit in the nucleus. My data supports a model whereby FKBP25 associates with the assembling large ribosomal subunit, where it is likely to chaperone protein-RNA interactions. / Graduate

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2007-2008. / La protéine multifonctionnelle p54nrb , enrichie dans un nouveau domaine nucléaire nommé paraspeckles, fait partie de complexes de transcription/épissage comprenant l'ARN polymérase II et son partenaire PSF. Des travaux récents effectués dans notre laboratoire montrent que p54nrb est phosphorylée en mitose (Proteau A., Blier S., Albert A.L., Lavoie S.B., Traish A. M. and Vincent M. (2005) J. Mol. Biol. 346, 1163-1172). La phosphorylation est une modification post-traductionnelle pouvant affecter la localisation cellulaire d'une protéine, ses interactions, sa dégradation et son activité. Pour étudier l'impact de la phosphorylation mitotique de p54nrb , sa localisation cellulaire a été comparée en interphase et en mitose par immunofluorescence indirecte. Les résultats ont montré que la phosphorylation ne semble pas affecter sa localisation aux paraspeckles en mitose. Ensuite, ses interactions dans le complexe transcription/épissage ont été vérifiées en mitose par immunoprécipitation et pulldown. Les résultats ont montré que la phosphorylation ne semble pas affecter le complexe p54nrb-PSF-ARN polymérase II en mitose in vitro. Des analyses biochimiques ont finalement montré que la phosphorylation de p54nrb ne semble pas non plus empêcher son association à la matrice nucléaire. L'étude antérieure, citée précédemment, a également montré que p54nrb est reconnue par la peptidyl-prolyl isomérase Pinl en mitose. La juglone, un inhibiteur enzymatique de Pinl, a été utilisée pour évaluer l'effet de l'interaction de Pinl sur le niveau de phosphorylation de p54nrb . Les résultats ont montré que l'inhibition de Pinl empêche la déphosphorylation de p54nrb à la fin de la mitose ainsi que celle de PSF, nouveau substrat de Pinl. La protéine Pinl pourrait réguler chaque membre du complexe p54nrb-PSF-ARN polymérase II à la reprise du cycle cellulaire.

<p>Peptide bond formation and translational termination on the ribosome have been simulated by molecular mechanics, free energy perturbation, empirical valence bond (MD/FEP/EVB) and automated docking methods. Recent X-ray crystallographic data is used here to calculate the entire free energy surface for the system complete with substrates, ribosomal groups, solvent molecules and ions. A reaction mechanism for peptide bond formation emerges that is found to be catalyzed by the ribosome, in agreement with kinetic data and activation entropy measurements. The results show a water mediated network of hydrogen bonds, capable of reducing the reorganization energy during peptidyl transfer. The predicted hydrogen bonds and the structure of the active site were later confirmed by new X-ray structures with proper transition states analogs. </p><p>Elongation termination on the ribosome is triggered by binding of a release factor (RF) protein followed by rapid release of the nascent peptide. The structure of the RF, bound to the ribosomal peptidyl transfer center (PTC), has not been resolved in atomic detail. Nor is the mechanism known, by which the hydrolysis proceeds. Using automated docking of a hepta-peptide RF fragment, containing the highly conserved GGQ motif, we identified a conformation capable of catalyzing peptide hydrolysis. The MD/FEP/EVB calculations also reproduce the slow spontaneous release when RF is absent, and rationalize available mutational data. The network of hydrogen bonds, the active site structure, and the reaction mechanism are found to be very similar for both peptidyl transfer and termination. </p><p>New structural data, placing a ribosomal protein (L27) in the PTC, motivated additional MD/FEP/EVB simulations to determine the effect of this protein on peptidyl transfer. The simulations predict that the protein N terminus interacts with the A-site substrate in a way that promotes binding. The catalytic effect of L27 in the ribosome, however, is shown to be marginal and it therefore seems valid to view the PTC as a ribozyme. Simulations with the model substrate puromycin (Pmn) predicts that protonation of the N terminus can reduce the rate of peptidyl transfer. This could explain the different pH-rate profiles measured for Pmn, compared to other substrates.</p>